U.S. patent number 11,319,531 [Application Number 16/652,941] was granted by the patent office on 2022-05-03 for transglutaminase variants.
This patent grant is currently assigned to Codexis, Inc.. The grantee listed for this patent is Codexis, Inc.. Invention is credited to Goutami Banerjee, Erika M. Milczek, Jeffrey C. Moore, Jovana Nazor, James Nicholas Riggins, Jie Yang, Xiyun Zhang.
United States Patent |
11,319,531 |
Nazor , et al. |
May 3, 2022 |
Transglutaminase variants
Abstract
The present invention provides engineered transglutamirase
enzymes, polynucleotides encoding the enzymes, compositions
comprising the enzymes, methods of producing these enzymes, and
methods of using the engineered transglutaminase enzymes.
Inventors: |
Nazor; Jovana (Milpitas,
CA), Yang; Jie (Foster City, CA), Banerjee; Goutami
(Hayward, CA), Zhang; Xiyun (Fremont, CA), Riggins; James
Nicholas (San Francisco, CA), Milczek; Erika M. (New
York, NY), Moore; Jeffrey C. (Westfield, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Codexis, Inc. |
Redwood City |
CA |
US |
|
|
Assignee: |
Codexis, Inc. (Redwood City,
CA)
|
Family
ID: |
1000006279756 |
Appl.
No.: |
16/652,941 |
Filed: |
November 2, 2018 |
PCT
Filed: |
November 02, 2018 |
PCT No.: |
PCT/US2018/059049 |
371(c)(1),(2),(4) Date: |
April 01, 2020 |
PCT
Pub. No.: |
WO2019/094301 |
PCT
Pub. Date: |
May 16, 2019 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200263150 A1 |
Aug 20, 2020 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62582593 |
Nov 7, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
9/1044 (20130101); C12N 15/63 (20130101); C07K
14/62 (20130101); C12Y 203/02013 (20130101) |
Current International
Class: |
C12N
9/10 (20060101); C07K 14/62 (20060101); C12N
15/63 (20060101) |
References Cited
[Referenced By]
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WO |
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|
Primary Examiner: Holland; Paul J
Attorney, Agent or Firm: Codexis, Inc.
Parent Case Text
The present application is a national stage application filed under
35 USC .sctn.371 and claims priority to international application
to PCT International Application No. PCT/US18/59049, filed Nov. 2,
2018, which claims priority to U.S. Prov. Pat. Appln. Ser. No.
62/582,593, filed Nov. 7, 2017, which is incorporated by reference
in its entirety for all purposes.
Claims
We claim:
1. An engineered transglutaminase having at least 90% sequence
identity to the amino acid sequence of SEQ ID NO: 2 and having
transglutaminase activity, wherein said engineered transglutaminase
having at least 90% sequence identity to the amino acid sequence of
SEQ ID NO: 2 comprises a substitution at one or more positions
selected from positions 48, 203, 343, and 346, wherein said
positions are numbered with reference to the amino acid sequence of
SEQ ID NO: 2.
2. The engineered transglutaminase of claim 1, wherein said
engineered transglutaminase having at least 90% sequence identity
to the amino acid sequence of SEQ ID NO: 2 further comprises at
least one substitution or substitution set at one or more positions
selected from positions 48/67/70, 48/67/70/181/203/256,
48/67/70/181/256/345,
43/67/70/131/296/345/373,43/67/70/203/256/296/345,
48/67/70/203/256/345/354/373, 48/67/70/203/345, 48/67/70/256,
48/67/70/256/296/345/373, 48/67/203/256/296/373, 48/67/203/256/345,
48/70/170/203, 48/70/203/254/296/343, 48/70/203/256/345/373,
48/70/203/256/345, 48/70/203/373, 48/170/203,
48/170/203/254/296/346, 48/170/203/254/296/346/373,
48/170/203/254/346/373, 48/170/203/254/346, 48/170/203/296/343/346,
48/170/203/296/346/373, 48/170/203/343/346, 48/170/203/346,
48/170/203/346/373, 48/170/203/373, 48/170/254, 48/170/296,
48/170/296/343/346, 48/170/343/346, 48/181, 48/181/203/756/345,
48/181/203/345, 48/181/256/296/345, 48/181/296, 48/18 1/296/345,
48/203/254/296, 48/203/254/296/343/373, 48/203/254/296/346/373,
48/203/254/346, 48/203/254/346/373, 48/203/256, 48/203/256/296/345,
48/203/296/343/346/373, 48/203/296/343/373, 48/203/296/346,
48/203/296/346/373, 48/203/343/346/373, 48/203/345, 48/203/346/373,
48/254/296, 48/254/346, 48/256, 48/256/296, 48/256/296/345,
48/296/345, 48/296/373, 48/345/373, 67/256, 67/296/345,
68/74/190/215/346, 68/136/215/255/282/297/346, 68/136/215/297/346,
68/136/234, 68/158/174/234/282/297/346, 68/158/215/297/346,
68/215/297/346, 68/234, 68/282/297/346, 8/297/346,
74/136/174/282/346, 74/136/174/297/346, 74/136/346, 74/158/255/297,
74/255/346, 74/346, 136/158/190/215/255/297/346,
136/158/215/297/346, 136/174/215/255/282/297/346,
136/190/21.5/297/346, 136/215/234/282/297, 136/215/234/297/346,
136/215/297, 136/297/346, 158/215/255/346, 158/215/346,
170/203/254/296/343/346, 170/203/254/343/373, 170/203/343/346,
174/190/234/297/346, 174/215/234/297/346, 174/215/255/297/346,
174/282/297/346, 190/255/282/346, 190/297/346, 203/296,
215/255/297/346, 215/234/297/346, 215/255/297/346, 215/297,
215/297/346, 215/346, 234/255/346, 255/297/346, 255/346, 297/346,
and 343/346/373, wherein said positions are numbered with reference
to the amino add sequence of SEQ ID NO:2.
3. The engineered transglutaminase of claim 1, wherein said
engineered transglutaminase comprises the amino add sequence of SEQ
ID NO: 34 or 256.
4. The engineered transglutaminase of claim 1, wherein said
engineered transglutaminase comprises the amino add sequence of SEQ
ID NO: 256.
5. The engineered transglutaminase of claim 1, wherein said
engineered transglutaminase is capable of modifying a free amine in
insulin in the presence of a glutamine donor.
6. The engineered transglutaminase of claim 1, wherein said
engineered transglutaminase is capable of modifying a glutamine in
insulin in the presence of a iysine donor.
7. A method of modifying insulin comprising: providing insulin and
the engineered transglutaminase of claim 1, combining said insulin,
glutamine and said engineered transglutaminse under conditions such
that said insulin is modified.
8. A method of modifying insulin comprising: providing insulin and
the engineered transglutaminase of claim 1, combining said insulin,
lysine and one said engineered transglutaminse under conditions
such that said insulin is modified.
Description
FIELD OF THE INVENTION
The present invention provides engineered transglutaminase enzymes,
polynucleotides encoding the enzymes, compositions comprising the
enzymes, methods of producing these enzymes, and methods of using
the engineered transglutaminase enzymes.
REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
The official copy of the Sequence Listing is submitted concurrently
with the specification as an ASCII formatted text file via EFS-Web,
with a file name of "CX2-164USP1_ST25.txt", a creation date of Nov.
7, 2017, and a size of 1,896 kilobytes. The Sequence Listing filed
via EFS-Web is part of the specification and is incorporated in its
entirety by reference herein.
BACKGROUND OF THE INVENTION
Transglutaminases (TGase; EP
2.3.2.13)(R-gluaminyl-peptide-aminase-gamma-glutamyltransferase)
comprise an enzyme family that catalyze post-translational
modifications in proteins, producing covalent amide bonds between a
primary amine group in a polyamine or lysine (i.e., an amine donor)
and a gamma-carboxyamide group of the glutamyl residue of some
proteins and polypeptides (i.e., an amine acceptor). The result of
this enzymatic action include modification of the protein's
conformation and/or extensive conformation changes resulting from
the bonding of the same and different proteins to produce high
molecular weight conjugates. These enzymes find use various
applications, including in the food, cosmetic, textile, and
pharmaceutical industries.
SUMMARY OF THE INVENTION
The present invention provides engineered transglutaminase enzymes,
polynucleotides encoding the enzymes, compositions comprising the
enzymes, and methods of using the engineered transglutaminase
enzymes.
The present invention provides engineered transglutaminases having
at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more sequence identity to SEQ ID NO: 2, 6, 34, and/or 256. In some
embodiments, the engineered transglutaminases have at least 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to SEQ ID NO:6, and at least one substitution or
substitution set at one or more positions selected from positions
79, 101, 101/201/212/287, 101/201/285, 101/287, and 327, wherein
said positions are numbered with reference to SEQ ID NO:6. In some
embodiments, the engineered transglutaminases have at least 85%%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to SEQ ID NO:2, and at least one substitution or
substitution set at one or more positions selected from positions
48, 48/67/70, 48/67/70/181/203/256, 48/67/70/181/256/345,
48/67/70/181/296/345/373, 48/67/70/203/256/296/345,
48/67/70/203/256/345/354/373, 48/67/70/203/345, 48/67/70/256,
48/67/70/256/296/345/373, 48/67/203/256/296/373, 48/67/203/256/345,
48/70/170/203, 48/70/203/254/296/343, 48/70/203/256/345/373,
48/70/203/256/345, 48/70/203/373, 48/170/203,
48/170/203/254/296/346, 48/170/203/254/296/346/373,
48/170/203/254/346/373, 48/170/203/254/346, 48/170/203/296/343/346,
48/170/203/296/346/373, 48/170/203/343/346, 48/170/203/346,
48/170/203/346/373, 48/170/203/373, 48/170/254, 48/170/296,
48/170/296/343/346, 48/170/343/346, 48/181, 48/181/203/256/345,
48/181/203/345, 48/181/256/296/345, 48/181/296, 48/181/296/345,
48/203, 48/203/254/296, 48/203/254/296/343/373,
48/203/254/296/346/373, 48/203/254/346, 48/203/254/346/373,
48/203/256, 48/203/256/296/345, 48/203/296/343/346/373,
48/203/296/343/373, 48/203/296/346, 48/203/296/346/373,
48/203/343/346, 48/203/343/346/373, 48/203/345, 48/203/346,
48/203/346/373, 48/254/296, 48/254/346, 48/256, 48/256/296,
48/256/296/345, 48/296/345, 48/296/373, 48/343/346, 48/345/373,
67/256, 67/296/345, 68/74/190/215/346, 68/136/215/255/282/297/346,
68/136/215/297/346, 68/136/234, 68/158/174/234/282/297/346,
68/158/215/297/346, 68/215/297/346, 68/234, 68/282/297/346,
68/297/346, 74/136/174/282/346, 74/136/174/297/346, 74/136/346,
74/158/255/297, 74/255/346, 74/346, 136/158/190/215/255/297/346,
136/158/215/297/346, 136/174/215/255/282/297/346,
136/190/215/297/346, 136/215/234/282/297, 136/215/234/297/346,
136/215/297, 136/297/346, 158/215/255/346, 158/215/346,
170/203/254/296/343/346, 170/203/254/343/373, 170/203/343/346,
174/190/234/297/346, 174/215/234/297/346, 174/215/255/297/346,
174/282/297/346, 190/255/282/346, 190/297/346, 203/296, 203/343,
203/343/346, 203/346, 215/255/297/346, 215/234/297/346,
215/255/297/346, 215/297, 215/297/346, 215/346, 234/255/346,
255/297/346, 255/346, 297/346, 343/346/373, and 346, wherein said
positions are numbered with reference to SEQ ID NO:2. In some
additional embodiments, the engineered transglutaminases have at
least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
sequence identity to SEQ ID NO: 2, and at least one substitution or
substitution set at one or more positions selected from
33/67/70/181/203/256/296/373, 36/48/203/254/346,
48/67/70/181/203/256/296/373, 48/67/70/203/256/296/373,
48/67/181/203/256/296/373, 48/67/181/203/256/373,
48/67/181/256/296, 48/67/203/256/296/373/378, 48/67/203/256/373,
48/67/203/296/373, 48/67/256/296/373, 48/70/181/203/256/296/373,
48/70/181/203/256/373, 48/70/181/203/296/373,
48/70/203/256/296/373, 48/70/203/256/373, 48/70/203/296,
48/70/203/296/373, 48/70/203/373, 48/70/256/296/373, 48/70/296/373,
48/176/203/254/346/373, 48/181/203/256/296/373, 48/181/203/256/373,
48/181/203/296, 48/181/203/373, 48/181/256/296/373, 48/203/254,
48/203/254/343, 48/203/254/343/346/373, 48/203/254/343/355/373,
48/203/254/343/373, 48/203/254/346/373, 48/203/254/373,
48/203/256/296, 48/203/256/296/373, 48/203/256/373, 48/203/296/373,
48/203/296/373/374, 48/203/343/373, 48/203/373, 48/254,
48/254/343/346/373, 48/254/343/373, 48/254/346/373, 48/254/373,
48/256/296/373, 48/256/373, 48/373, 67/70/181/203/256/296/373,
67/70/181/256/296/373, 67/70/181/373, 67/181/203/256/296,
67/181/203/256/296/373, 67/181/203/256/373, 67/203/256/296/373,
67/256/296/373, 70/181/203/256/296/373, 70/181/203/296/373, 70/203,
70/203/256/296/373, 70/203/256/373, 70/203/296/373,
74/136/215/234/282/297/346, 74/136/215/234/282/346,
74/136/215/234/297, 74/136/215/234/297/343/346,
74/136/215/234/297/346, 74/136/215/234/346, 74/136/215/282/297/346,
74/136/215/282/346, 74/136/215/297/346, 74/136/215/346,
74/136/234/282/297/346, 74/136/234/346, 74/136/282/297/346, 74/215,
74/215/234/282/297/346, 74/215/282/297/346, 74/215/346,
136/215/234/282/297/346, 136/215/282/297, 136/215/282/297/346,
136/215/282/346, 136/215/297/346, 136/215/346, 136/234/297,
136/234/297/346, 136/234/346, 136/282/297, 181/203/256,
181/203/256/296, 181/203/256/296/373, 181/203/256/373,
181/203/296/373, 181/203/373, 181/256/296/373, 181/296,
203/224/254/373, 203/254, 203/254/343/346/373, 203/254/343/373,
203/254/346, 203/254/346/373, 203/254/373, 203/346/373, 203/373,
203/209/256/373, 203/256, 203/256/296, 203/256/296/320/373,
203/256/296/373, 203/256/296/373/386, 203/256/373, 203/296/373,
203/373, 215/234/282/297/346, 215/234/282/346, 215/234/346,
234/282/346, 254, 254/346, 254/346/373, 254/373, 256/296,
256/296/373, 256/373, 282/297/346, 343/373, and 373, wherein said
positions are numbered with reference to SEQ ID NO: 2. In some
further embodiments, the engineered transglutaminases have at least
850/%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%6 or more
sequence identity to SEQ ID NO: 34, and at least one substitution
or substitution set at one or more positions selected from 48/49,
49, 50, 50, 331, 291, 292, 330, and 331, wherein said positions are
numbered with reference to SEQ ID NO: 34. In yet some additional
embodiments, the engineered transglutaminases have at least 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence
identity to SEQ ID NO: 256, and at least one substitution or
substitution set at one or more positions selected from
27/48/67/70/74/234/256/282/346/373,
27/48/67/70/136/203/215/256/282/346/373, 27/48/67/70/346/373,
27/48/67/74/203/256/346/373, 27/67/234/296/373, 45/287/328/333,
45/292/328, 48, 48/284/292/333, 48/287/292/297, 48/287/297/328/333,
48/292, 48/292/297, 48/49/50/292/331, 48/49/50/292, 48/49/50/331,
48/49/330/331, 48/49/50/349, 48/49/50/291/292/331,
48/49/50/292/331, 48/67/70/203/215/234/256/346,
48/67/70/234/256/282/297/346, 48/67/70/346,
48/67/74/203/234/256/282/346/373, 48/67/74/234/297/346/373,
48/67/74/346, 48/67/203/346/373, 48/67/234/256/297/346/373,
48/67/234/256/346/373, 48/67/215/282/297/346/373, 48/67/346/373,
48/70/74/297/346/373, 48/70/203/215/256/282/346/373,
48/70/215/234/256/346/373, 48/74/203/234/256/346/373,
48/74/234/256/297/346/373, 48/136/256/346/373,
48/203/234/256/297/346/373, 48/203/234/256/346/373,
48/203/234/346/373, 48/203/296/373, 48/215/234/346/373,
48/215/346/373, 48/234/256/296/346/373, 48/234/256/346/373,
48/256/373, 49/50/292/331, 49/50/292/331/349, 49/50/331,
49/50/331/349, 50, 67/70/74/136/203/215/256/346/373,
67/70/74/203/215/234/346/373, 67/70/74/215/234/297/346/373,
67/70/74/215/256/373, 67/70/136/203/297/346/373,
67/70/203/215/256/346/373, 67/70/203/373, 67/70/215, 67/74/136,
67/74/203/234/256, 67/74/215/256/297/346/373, 67/74/215/346/373,
67/74/256/346/373, 67/136/203/215/256/346/373,
67/136/203/256/346/373, 67/203/234/256/346/373, 67/203/297/346/373,
67/215/234/297/346/373, 67/297/346, 70/74/203/215/346/373, 136,
136/346/373, 203/234/346, 203/234/346/373, 203/373, 234/282, 287,
234/346/373, 287/292, 287/292/295/297, 287/292/297, 287/295/297,
287/330/333, 292, 292/297, 292/330/331, 292/330/331, 292/331,
292/331/349, 292/349, 295, 295/297/333, 297/328, 297/373, 328/333,
330, 330/331, 331, 331/349, 333, 346/373, and 373, wherein said
positions are numbered with reference to SEQ ID NO: 256. In some
embodiments, the engineered transglutaminases comprise a
polypeptide sequence comprising a sequence having at least 90%
sequence identity to SEQ ID NO:2, 6, 34, and/or 256. In some
alternative embodiments, the engineered transglutaminases comprise
a polypeptide sequence comprising a sequence having at least 95%
sequence identity to SEQ ID NO:2, 6, 34, and/or 256. In yet some
additional embodiments, the engineered transglutaminases comprise a
polypeptide sequence set forth in SEQ ID NO:2, 6, 34, or 256. In
yet some additional embodiments, the engineered transglutaminases
comprise a polypeptide sequence encoding a variant provided in
Table 8.1, 9.1, 9.2, 10.1, and/or 11.1. In some further
embodiments, the engineered transglutaminase comprises a
polypeptide sequence selected from the even-numbered sequences set
from in SEQ ID NOS: 4 to 756.
The present invention also provides engineered polynucleotide
sequences encoding the engineered transglutaminases provided
herein. In some embodiments, the engineered polynucleotide
sequences comprise polynucleotide sequences that are at least 85%,
90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or more identical
to a sequence selected from the odd-numbered sequences set forth in
SEQ ID NOS: 3 to 755. The present invention also provides vectors
comprising the engineered polynucleotide sequences provided herein.
In some embodiments, the vectors further comprise at least one
control sequence. The present invention also provides host cells
comprising the vectors provided herein.
The present invention also provides methods for producing the
engineered transglutaminases provided herein, comprising culturing
a host cell under conditions that at least one engineered
transglutaminase is produced by said host cell. In some
embodiments, the host cell produces an engineered transglutaminase.
In some embodiments, the methods further comprise the step of
recovering said engineered transglutaminase produced by said host
cell.
The present invention also provides engineered transglutaminases
capable of modifying a free amine in insulin in the presence of a
glutamine donor. In some embodiments, the engineered
transglutaminases provided herein are capable of modifying a
glutamine in insulin in the presence of a lysine donor.
The present invention also provides methods of modifying insulin
comprising: providing insulin and at least one engineered
transglutaminase provided herein, combining said insulin,
glutamine, and at least one engineered transglutaminase under
conditions such that said insulin is modified. The present
invention also provides methods of modifying insulin comprising:
providing insulin and at least one engineered transglutaminase
provided herein, combining said insulin, lysine, and at least one
engineered transglutaminase under conditions such that said insulin
is modified.
DESCRIPTION OF THE INVENTION
The present invention provides engineered transglutaminase enzymes,
polynucleotides encoding the enzymes, compositions comprising the
enzymes, and methods of using the engineered transglutaminase
enzymes.
The transglutaminase variants provided herein are engineered from
the Streptomyces mobaraensis transglutaminase of SEQ ID NO:2, in
which various modifications have been introduced to generate
improved enzymatic properties as described in detail below.
For the descriptions provided herein, the use of the singular
includes the plural (and vice versa) unless specifically stated
otherwise. For instance, the singular forms "a", "an" and "the"
include plural referents unless the context clearly indicates
otherwise. Similarly, "comprise." "comprises," "comprising"
"include," "includes," and "including" are interchangeable and not
intended to be limiting.
It is to be further understood that where descriptions of various
embodiments use the term "comprising," those skilled in the art
would understand that in some specific instances, an embodiment can
be alternatively described using language "consisting essentially
of" or "consisting of."
Both the foregoing general description, including the drawings, and
the following detailed description are exemplary and explanatory
only and are not restrictive of this disclosure. Moreover, the
section headings used herein are for organizational purposes only
and not to be construed as limiting the subject matter
described.
Definitions
As used herein, the following terms are intended to have the
following meanings. In reference to the present disclosure, the
technical and scientific terms used in the descriptions herein will
have the meanings commonly understood by one of ordinary skill in
the art, unless specifically defined otherwise. Accordingly, the
following terms are intended to have the following meanings. In
addition, all patents and publications, including all sequences
disclosed within such patents and publications, referred to herein
are expressly incorporated by reference.
Unless otherwise indicated, the practice of the present invention
involves conventional techniques commonly used in molecular
biology, fermentation, microbiology, and related fields, which are
known to those of skill in the art. Unless defined otherwise
herein, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. Although any methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, the
preferred methods and materials are described. Indeed, it is
intended that the present invention not be limited to the
particular methodology, protocols, and reagents described herein,
as these may vary, depending upon the context in which they are
used. The headings provided herein are not limitations of the
various aspects or embodiments of the present invention that can be
had by reference to the specification as a whole. Accordingly, the
terms defined below are more fully defined by reference to the
specification as a whole.
Nonetheless, in order to facilitate understanding of the present
invention, a number of terms are defined below. Numeric ranges are
inclusive of the numbers defining the range. Thus, every numerical
range disclosed herein is intended to encompass every narrower
numerical range that falls within such broader numerical range, as
if such narrower numerical ranges were all expressly written
herein. It is also intended that every maximum (or minimum)
numerical limitation disclosed herein includes every lower (or
higher) numerical limitation, as if such lower (or higher)
numerical limitations were expressly written herein.
As used herein, the term "comprising" and its cognates are used in
their inclusive sense (i.e., equivalent to the term "including" and
its corresponding cognates).
As used herein and in the appended claims, the singular "a", "an"
and "the" include the plural reference unless the context clearly
dictates otherwise. Thus, for example, reference to a "host cell"
includes a plurality of such host cells.
Unless otherwise indicated, nucleic acids are written left to right
in 5' to 3' orientation and amino acid sequences are written left
to right in amino to carboxy orientation, respectively.
As used herein, the terms "protein," "polypeptide," and "peptide"
are used interchangeably herein to denote a polymer of at least two
amino acids covalently linked by an amide bond, regardless of
length or post-translational modification (e.g., glycosylation,
phosphorylation, lipidation, myristilation, ubiquitination, etc.).
Included within this definition are D- and L-amino acids, and
mixtures of D- and L-amino acids.
As used herein, "polynucleotide" and "nucleic acid" refer to two or
more nucleosides that are covalently linked together. The
polynucleotide may be wholly comprised ribonucleosides (i.e., an
RNA), wholly comprised of 2' deoxyribonucleotides (i.e., a DNA) or
mixtures of ribo- and 2' deoxyribonucleosides. While the
nucleosides will typically be linked together via standard
phosphodiester linkages, the polynucleotides may include one or
more non-standard linkages. The polynucleotide may be
single-stranded or double-stranded, or may include both
single-stranded regions and double-stranded regions. Moreover,
while a polynucleotide will typically be composed of the naturally
occurring encoding nucleobases (i.e., adenine, guanine, uracil,
thymine, and cytosine), it may include one or more modified and/or
synthetic nucleobases (e.g., inosine, xanthine, hypoxanthine,
etc.). Preferably, such modified or synthetic nucleobases will be
encoding nucleobases.
As used herein, "hybridization stringency" relates to hybridization
conditions, such as washing conditions, in the hybridization of
nucleic acids. Generally, hybridization reactions are performed
under conditions of lower stringency, followed by washes of varying
but higher stringency.
The term "moderately stringent hybridization" refers to conditions
that permit target-DNA to bind a complementary nucleic acid that
has about 60% identity, preferably about 75% identity, about 85%
identity to the target DNA; with greater than about 90% identity to
target-polynucleotide. Exemplary moderately stringent conditions
are conditions equivalent to hybridization in 50% formamide,
5.times. Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree.
C., followed by washing in 0.2.times.SSPE, 0.2% SDS, at 42.degree.
C. "High stringency hybridization" refers generally to conditions
that are about 10.degree. C. or less from the thermal melting
temperature T, as determined under the solution condition for a
defined polynucleotide sequence. In some embodiments, a high
stringency condition refers to conditions that permit hybridization
of only those nucleic acid sequences that form stable hybrids in
0.018M NaCl at 65.degree. C. (i.e., if a hybrid is not stable in
0.018M NaCl at 65.degree. C., it will not be stable under high
stringency conditions, as contemplated herein). High stringency
conditions can be provided, for example, by hybridization in
conditions equivalent to 50% formamide, 5.times. Denhart's
solution, 5: SSPE, 0.2% SDS at 42.degree. C., followed by washing
in 0.1.times.SSPE, and 0.1% SDS at 65.degree. C. Another high
stringency condition is hybridizing in conditions equivalent to
hybridizing in 5.times.SSC containing 0.1% (w:v) SDS at 65.degree.
C. and washing in 0.1.times.SSC containing 0.1% SDS at 65.degree.
C. Other high stringency hybridization conditions, as well as
moderately stringent conditions, are known to those of skill in the
art.
As used herein, "coding sequence" refers to that portion of a
nucleic acid (e.g., a gene) that encodes an amino acid sequence of
a protein.
As used herein, "codon optimized" refers to changes in the codons
of the polynucleotide encoding a protein to those preferentially
used in a particular organism such that the encoded protein is
efficiently expressed in the organism of interest. In some
embodiments, the polynucleotides encoding the transglutaminase
enzymes may be codon optimized for optimal production from the host
organism selected for expression. Although the genetic code is
degenerate in that most amino acids are represented by several
codons, called "synonyms" or "synonymous" codons, it is well known
that codon usage by particular organisms is nonrandom and biased
towards particular codon triplets. This codon usage bias may be
higher in reference to a given gene, genes of common function or
ancestral origin, highly expressed proteins versus low copy number
proteins, and the aggregate protein coding regions of an organism's
genome. In some embodiments, the polynucleotides encoding the
transglutaminase enzymes may be codon optimized for optimal
production from the host organism selected for expression.
As used herein, "preferred, optimal, high codon usage bias codons"
refers interchangeably to codons that are used at higher frequency
in the protein coding regions than other codons that code for the
same amino acid. The preferred codons may be determined in relation
to codon usage in a single gene, a set of genes of common function
or origin, highly expressed genes, the codon frequency in the
aggregate protein coding regions of the whole organism, codon
frequency in the aggregate protein coding regions of related
organisms, or combinations thereof. Codons whose frequency
increases with the level of gene expression are typically optimal
codons for expression. A variety of methods are known for
determining the codon frequency (e.g, codon usage, relative
synonymous codon usage) and codon preference in specific organisms,
including multivariate analysis, for example, using cluster
analysis or correspondence analysis, and the effective number of
codons used in a gene (See e.g., GCG CodonPreference, Genetics
Computer Group Wisconsin Package; CodonW, John Peden, University of
Nottingham; Mclnerney, Bioinform., 14:372-73 [1998]; Stenico et
al., Nucleic Acids Res., 222:437-46 [1994] and Wright. Gene
87:23-29 [1990]). Codon usage tables are available for a growing
list of organisms (See e.g., Wada et al., Nucleic Acids Res.,
20:2111-2118 [1992]; Nakamura et al., Nucl. Acids Res., 28:292
[2000]; Duret, et al., supra: Henaut and Danchin, "Escherichia coli
and Salmonella." Neidhardt, et al. (eds.), ASM Press, Washington
D.C., [1996], p. 2047-2066. The data source for obtaining codon
usage may rely on any available nucleotide sequence capable of
coding for a protein. These data sets include nucleic acid
sequences actually known to encode expressed proteins (e.g.,
complete protein coding sequences-CDS), expressed sequence tags
(ESTS), or predicted coding regions of genomic sequences (See e.g.,
Uberbacher, Meth. Enzymol., 266:259-281 [1996]; Tiwari et al.,
Comput. Appl. Biosci., 13:263-270 [1997]).
As used herein, "control sequence" is defined herein to include all
components, which are necessary or advantageous for the expression
of a polynucleotide and/or polypeptide of the present invention.
Each control sequence may be native or foreign to the
polynucleotide of interest. Such control sequences include, but are
not limited to, a leader, polyadenylation sequence, propeptide
sequence, promoter, signal peptide sequence, and transcription
terminator.
As used herein, "operably linked" is defined herein as a
configuration in which a control sequence is appropriately placed
(i.e., in a functional relationship) at a position relative to a
polynucleotide of interest such that the control sequence directs
or regulates the expression of the polynucleotide and/or
polypeptide of interest.
As used herein, "promoter sequence" refers to a nucleic acid
sequence that is recognized by a host cell for expression of a
polynucleotide of interest, such as a coding sequence. The control
sequence may comprise an appropriate promoter sequence. The
promoter sequence contains transcriptional control sequences, which
mediate the expression of a polynucleotide of interest. The
promoter may be any nucleic acid sequence which shows
transcriptional activity in the host cell of choice including
mutant, truncated, and hybrid promoters, and may be obtained from
genes encoding extracellular or intracellular polypeptides either
homologous or heterologous to the host cell.
As used herein, "naturally occurring" and "wild-type" refers to the
form found in nature. For example, a naturally occurring or
wild-type polypeptide or polynucleotide sequence is a sequence
present in an organism that can be isolated from a source in nature
and which has not been intentionally modified by human
manipulation.
As used herein, "non-naturally occurring," "engineered," and
"recombinant" when used in the present disclosure with reference to
(e.g., a cell, nucleic acid, or polypeptide), refers to a material,
or a material corresponding to the natural or native form of the
material, that has been modified in a manner that would not
otherwise exist in nature. In some embodiments the material is
identical to naturally occurring material, but is produced or
derived from synthetic materials and/or by manipulation using
recombinant techniques. Non-limiting examples include, among
others, recombinant cells expressing genes that are not found
within the native (non-recombinant) form of the cell or express
native genes that are otherwise expressed at a different level.
As used herein, "percentage of sequence identity," "percent
identity," and "percent identical" refer to comparisons between
polynucleotide sequences or polypeptide sequences, and are
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide or
polypeptide sequence in the comparison window may comprise
additions or deletions (i.e., gaps) as compared to the reference
sequence for optimal alignment of the two sequences. The percentage
is calculated by determining the number of positions at which
either the identical nucleic acid base or amino acid residue occurs
in both sequences or a nucleic acid base or amino acid residue is
aligned with a gap to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity. Determination of
optimal alignment and percent sequence identity is performed using
the BLAST and BLAST 2.0 algorithms (See e.g., Altschul et al., J.
Mol. Biol. 215: 403-410 [1990] and Altschul et al., Nucl. Acids
Res. 3389-3402 [1977]). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information website.
Briefly, the BLAST analyses involve first identifying high scoring
sequence pairs (HSPs) by identifying short words of length Win the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as, the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value: the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4, and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength
(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix
(See e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 [1989]).
Numerous other algorithms are available and known in the art that
function similarly to BLAST in providing percent identity for two
sequences. Optimal alignment of sequences for comparison can be
conducted using any suitable method known in the art (e.g., by the
local homology algorithm of Smith and Waterman, Adv. Appl. Math.
2:482 [1981]; by the homology alignment algorithm of Needleman and
Wunsch, J. Mol. Biol. 48:443 [1970]; by the search for similarity
method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444
[1988]; and/or by computerized implementations of these algorithms
[GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software
Package]), or by visual inspection, using methods commonly known in
the art. Additionally, determination of sequence alignment and
percent sequence identity can employ the BESTFIT or GAP programs in
the GCG Wisconsin Software package (Accelrys, Madison Wis.), using
the default parameters provided.
As used herein, "substantial identity" refers to a polynucleotide
or polypeptide sequence that has at least 80 percent sequence
identity, at least 85 percent identity and 89 to 95 percent
sequence identity, more usually at least 99 percent sequence
identity as compared to a reference sequence over a comparison
window of at least 20 residue positions, frequently over a window
of at least 30-50 residues, wherein the percentage of sequence
identity is calculated by comparing the reference sequence to a
sequence that includes deletions or additions which total 20
percent or less of the reference sequence over the window of
comparison. In specific embodiments applied to polypeptides, the
term "substantial identity" means that two polypeptide sequences,
when optimally aligned, such as by the programs GAP or BESTFIT
using default gap weights, share at least 80 percent sequence
identity, preferably at least 89 percent sequence identity, at
least 95 percent sequence identity or more (e.g., 99 percent
sequence identity). In some preferred embodiments, residue
positions that are not identical differ by conservative amino acid
substitutions.
As used herein, "reference sequence" refers to a defined sequence
to which another sequence is compared. A reference sequence may be
a subset of a larger sequence, for example, a segment of a
full-length gene or polypeptide sequence. Generally, a reference
sequence is at least 20 nucleotide or amino acid residues in
length, at least 25 residues in length, at least 50 residues in
length, or the full length of the nucleic acid or polypeptide.
Since two polynucleotides or polypeptides may each (1) comprise a
sequence (i.e., a portion of the complete sequence) that is similar
between the two sequences, and (2) may further comprise a sequence
that is divergent between the two sequences, sequence comparisons
between two (or more) polynucleotides or polypeptide are typically
performed by comparing sequences of the two polynucleotides over a
comparison window to identify and compare local regions of sequence
similarity. The term "reference sequence" is not intended to be
limited to wild-type sequences, and can include engineered or
altered sequences. For example, in some embodiments, a "reference
sequence" can be a previously engineered or altered amino acid
sequence.
As used herein, "comparison window" refers to a conceptual segment
of at least about 20 contiguous nucleotide positions or amino acids
residues wherein a sequence may be compared to a reference sequence
of at least 20 contiguous nucleotides or amino acids and wherein
the portion of the sequence in the comparison window may comprise
additions or deletions (i.e., gaps) of 20 percent or less as
compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The comparison window can be longer than 20 contiguous residues,
and includes, optionally 30, 40, 50, 100, or longer windows.
As used herein, "corresponding to," "reference to," and "relative
to" when used in the context of the numbering of a given amino acid
or polynucleotide sequence refers to the numbering of the residues
of a specified reference sequence when the given amino acid or
polynucleotide sequence is compared to the reference sequence. In
other words, the residue number or residue position of a given
polymer is designated with respect to the reference sequence rather
than by the actual numerical position of the residue within the
given amino acid or polynucleotide sequence. For example, a given
amino acid sequence, such as that of an engineered
transglutaminase, can be aligned to a reference sequence by
introducing gaps to optimize residue matches between the two
sequences. In these cases, although the gaps are present, the
numbering of the residue in the given amino acid or polynucleotide
sequence is made with respect to the reference sequence to which it
has been aligned. As used herein, a reference to a residue
position, such as "Xn" as further described below, is to be
construed as referring to "a residue corresponding to", unless
specifically denoted otherwise. Thus, for example, "X94" refers to
any amino acid at position 94 in a polypeptide sequence.
As used herein, "improved enzyme property" refers to a
transglutaminase that exhibits an improvement in any enzyme
property as compared to a reference transglutaminase. For the
engineered transglutaminase polypeptides described herein, the
comparison is generally made to the wild-type transglutaminase
enzyme, although in some embodiments, the reference
transglutaminase is another improved engineered transglutaminase.
Enzyme properties for which improvement is desirable include, but
are not limited to, enzymatic activity (which can be expressed in
terms of percent conversion of the substrate at a specified
reaction time using a specified amount of transglutaminase),
chemoselectivity, thermal stability, solvent stability, pH activity
profile, cofactor requirements, refractoriness to inhibitors (e.g.,
product inhibition), stereospecificity, and stereoselectivity
(including enantioselectivity).
As used herein, "increased enzymatic activity" refers to an
improved property of the engineered transglutaminase polypeptides,
which can be represented by an increase in specific activity (e.g.,
product produced/time/weight protein) or an increase in percent
conversion of the substrate to the product (e.g., percent
conversion of starting amount of substrate to product in a
specified time period using a specified amount of transglutaminase)
as compared to the reference transglutaminase enzyme. Exemplary
methods to determine enzyme activity are provided in the Examples.
Any property relating to enzyme activity may be affected, including
the classical enzyme properties of K.sub.m, V.sub.max or k.sub.cat,
changes of which can lead to increased enzymatic activity.
Improvements in enzyme activity can be from about 1.5 times the
enzymatic activity of the corresponding wild-type transglutaminase
enzyme, to as much as 2 times, 5 times, 10 times, 20 times, 25
times, 50 times, 75 times, 100 times, or more enzymatic activity
than the naturally occurring transglutaminase or another engineered
transglutaminase from which the transglutaminase polypeptides were
derived. In specific embodiments, the engineered transglutaminase
enzyme exhibits improved enzymatic activity in the range of 1.5 to
50 times, 1.5 to 100 times greater than that of the parent
transglutaminase enzyme. It is understood by the skilled artisan
that the activity of any enzyme is diffusion limited such that the
catalytic turnover rate cannot exceed the diffusion rate of the
substrate, including any required cofactors. The theoretical
maximum of the diffusion limit, or k.sub.cat/K.sub.m, is generally
about 10.sup.8 to 10.sup.9 (M.sup.-1 s.sup.-1). Hence, any
improvements in the enzyme activity of the transglutaminase will
have an upper limit related to the diffusion rate of the substrates
acted on by the transglutaminase enzyme.
Transglutaminase activity can be measured by any one of standard
assays available in the art (e.g., hydroxymate assays). Comparisons
of enzyme activities are made using a defined preparation of
enzyme, a defined assay under a set condition, and one or more
defined substrates, as further described in detail herein.
Generally, when lysates are compared, the numbers of cells and the
amount of protein assayed are determined as well as use of
identical expression systems and identical host cells to minimize
variations in amount of enzyme produced by the host cells and
present in the lysates.
As used herein, "increased enzymatic activity" and "increased
activity" refer to an improved property of an engineered enzyme,
which can be represented by an increase in specific activity (e.g.,
product produced/time/weight protein) or an increase in percent
conversion of the substrate to the product (e.g., percent
conversion of starting amount of substrate to product in a
specified time period using a specified amount of transglutaminase)
as compared to a reference enzyme as described herein. Any property
relating to enzyme activity may be affected, including the
classical enzyme properties of K.sub.m, V.sub.max, or k.sub.cat,
changes of which can lead to increased enzymatic activity.
Comparisons of enzyme activities are made using a defined
preparation of enzyme, a defined assay under a set condition, and
one or more defined substrates, as further described in detail
herein. Generally, when enzymes in cell lysates are compared, the
numbers of cells and the amount of protein assayed are determined
as well as use of identical expression systems and identical host
cells to minimize variations in amount of enzyme produced by the
host cells and present in the lysates.
As used herein, "conversion" refers to the enzymatic transformation
of a substrate to the corresponding product.
As used herein "percent conversion" refers to the percent of the
substrate that is converted to the product within a period of time
under specified conditions. Thus, for example, the "enzymatic
activity" or "activity" of a transglutaminase polypeptide can be
expressed as "percent conversion" of the substrate to the
product.
As used herein, "chemoselectivity" refers to the preferential
formation in a chemical or enzymatic reaction of one product over
another.
As used herein, "thermostable" and "thermal stable" are used
interchangeably to refer to a polypeptide that is resistant to
inactivation when exposed to a set of temperature conditions (e.g.,
40-80.degree. C.) for a period of time (e.g., 0.5-24 hrs) compared
to the untreated enzyme, thus retaining a certain level of residual
activity (e.g., more than 60% to 80%) after exposure to elevated
temperatures.
As used herein, "solvent stable" refers to the ability of a
polypeptide to maintain similar activity (e.g., more than e.g., 60%
to 80%) after exposure to varying concentrations (e.g., 5-99%) of
solvent (e.g., isopropyl alcohol, tetrahydrofuran,
2-methyltetrahydrofuran, acetone, toluene, butylacetate, methyl
tert-butylether, etc.) for a period of time (e.g., 0.5-24 hrs)
compared to the untreated enzyme.
As used herein, "pH stable" refers to a transglutaminase
polypeptide that maintains similar activity (e.g., more than 60% to
80%) after exposure to high or low pH (e.g., 4.5-6 or 8 to 12) for
a period of time (e.g., 0.5-24 hrs) compared to the untreated
enzyme.
As used herein, "thermo- and solvent stable" refers to a
transglutaminase polypeptide that is both thermostable and solvent
stable.
As used herein, "hydrophilic amino acid or residue" refers to an
amino acid or residue having a side chain exhibiting a
hydrophobicity of less than zero according to the normalized
consensus hydrophobicity scale of Eisenberg et al., (Eisenberg et
al., J. Mol. Biol., 179:125-142 [1984]).
Genetically encoded hydrophilic amino acids include L-Thr (T),
L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D),
L-Lys (K) and L-Arg (R).
As used herein, "acidic amino acid or residue" refers to a
hydrophilic amino acid or residue having a side chain exhibiting a
pK value of less than about 6 when the amino acid is included in a
peptide or polypeptide. Acidic amino acids typically have
negatively charged side chains at physiological pH due to loss of a
hydrogen ion. Genetically encoded acidic amino acids include L-Glu
(E) and L-Asp (D).
As used herein, "basic amino acid or residue" refers to a
hydrophilic amino acid or residue having a side chain exhibiting a
pK value of greater than about 6 when the amino acid is included in
a peptide or polypeptide. Basic amino acids typically have
positively charged side chains at physiological pH due to
association with hydronium ion. Genetically encoded basic amino
acids include L-Arg (R) and L-Lys (K).
As used herein, "polar amino acid or residue" refers to a
hydrophilic amino acid or residue having a side chain that is
uncharged at physiological pH, but which has at least one bond in
which the pair of electrons shared in common by two atoms is held
more closely by one of the atoms. Genetically encoded polar amino
acids include L-Asn (N), L-Gln (Q), L-Ser (S) and L-Thr (T).
As used herein, "hydrophobic amino acid or residue" refers to an
amino acid or residue having a side chain exhibiting a
hydrophobicity of greater than zero according to the normalized
consensus hydrophobicity scale of Eisenberg et al., (Eisenberg et
al., J. Mol. Biol., 179:125-142 [1984]). Genetically encoded
hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F),
L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr
(Y).
As used herein, "aromatic amino acid or residue" refers to a
hydrophilic or hydrophobic amino acid or residue having a side
chain that includes at least one aromatic or heteroaromatic
ring.
Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr
(Y) and L-Trp (W). Although owing to the pKa of its heteroaromatic
nitrogen atom L-His (H) it is sometimes classified as a basic
residue, or as an aromatic residue as its side chain includes a
heteroaromatic ring, herein histidine is classified as a
hydrophilic residue or as a "constrained residue" (see below).
As used herein, "constrained amino acid or residue" refers to an
amino acid or residue that has a constrained geometry. Herein,
constrained residues include L-Pro (P) and L-His (H). Histidine has
a constrained geometry because it has a relatively small imidazole
ring. Proline has a constrained geometry because it also has a five
membered ring.
As used herein, "non-polar amino acid or residue" refers to a
hydrophobic amino acid or residue having a side chain that is
uncharged at physiological pH and which has bonds in which the pair
of electrons shared in common by two atoms is generally held
equally by each of the two atoms (i.e., the side chain is not
polar). Genetically encoded non-polar amino acids include L-Gly
(G), L-Leu (L), L-Val (V). L-Ile (I), L-Met (M) and L-Ala (A).
As used herein, "aliphatic amino acid or residue" refers to a
hydrophobic amino acid or residue having an aliphatic hydrocarbon
side chain. Genetically encoded aliphatic amino acids include L-Ala
(A), L-Val (V), L-Leu (L) and L-Ile (I). It is noted that cysteine
(or "L-Cys" or "[C]") is unusual in that it can form disulfide
bridges with other L-Cys (C) amino acids or other sulfanyl- or
sulfhydryl-containing amino acids. The "cysteine-like residues"
include cysteine and other amino acids that contain sulfhydryl
moieties that are available for formation of disulfide bridges. The
ability of L-Cys (C) (and other amino acids with --SH containing
side chains) to exist in a peptide in either the reduced free --SH
or oxidized disulfide-bridged form affects whether L-Cys (C)
contributes net hydrophobic or hydrophilic character to a peptide.
While L-Cys (C) exhibits a hydrophobicity of 0.29 according to the
normalized consensus scale of Eisenberg (Eisenberg et al., 1984,
supra), it is to be understood that for purposes of the present
disclosure, L-Cys (C) is categorized into its own unique group.
As used herein, "small amino acid or residue" refers to an amino
acid or residue having a side chain that is composed of a total
three or fewer carbon and/or heteroatoms (excluding the
.alpha.-carbon and hydrogens). The small amino acids or residues
may be further categorized as aliphatic, non-polar, polar or acidic
small amino acids or residues, in accordance with the above
definitions. Genetically-encoded small amino acids include L-Ala
(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and
L-Asp (D).
As used herein, "hydroxyl-containing amino acid or residue" refers
to an amino acid containing a hydroxyl (--OH) moiety.
Genetically-encoded hydroxyl-containing amino acids include L-Ser
(S) L-Thr (T) and L-Tyr (Y).
As used herein, "amino acid difference" and "residue difference"
refer to a difference in the amino acid residue at a position of a
polypeptide sequence relative to the amino acid residue at a
corresponding position in a reference sequence. The positions of
amino acid differences generally are referred to herein as "Xn,"
where n refers to the corresponding position in the reference
sequence upon which the residue difference is based. For example, a
"residue difference at position X40 as compared to SEQ ID NO:2"
refers to a difference of the amino acid residue at the polypeptide
position corresponding to position 40 of SEQ ID NO:2. Thus, if the
reference polypeptide of SEQ ID NO:2 has a histidine at position
40, then a "residue difference at position X40 as compared to SEQ
ID NO:2" refers to an amino acid substitution of any residue other
than histidine at the position of the polypeptide corresponding to
position 40 of SEQ ID NO:2. In most instances herein, the specific
amino acid residue difference at a position is indicated as "XnY"
where "Xn" specified the corresponding position as described above,
and "Y" is the single letter identifier of the amino acid found in
the engineered polypeptide (i.e., the different residue than in the
reference polypeptide). In some instances, the present disclosure
also provides specific amino acid differences denoted by the
conventional notation "AnB", where A is the single letter
identifier of the residue in the reference sequence, "n" is the
number of the residue position in the reference sequence, and B is
the single letter identifier of the residue substitution in the
sequence of the engineered polypeptide. In some instances, a
polypeptide of the present disclosure can include one or more amino
acid residue differences relative to a reference sequence, which is
indicated by a list of the specified positions where residue
differences are present relative to the reference sequence. In some
embodiments, where more than one amino acid can be used in a
specific residue position of a polypeptide, the various amino acid
residues that can be used are separated by a "/" (e.g., X192A/G).
The present disclosure includes engineered polypeptide sequences
comprising one or more amino acid differences that include
either/or both conservative and non-conservative amino acid
substitutions. The amino acid sequences of the specific recombinant
carbonic anhydrase polypeptides included in the Sequence Listing of
the present disclosure include an initiating methionine (M) residue
(i.e., M represents residue position 1). The skilled artisan,
however, understands that this initiating methionine residue can be
removed by biological processing machinery, such as in a host cell
or in vitro translation system, to generate a mature protein
lacking the initiating methionine residue, but otherwise retaining
the enzyme's properties. Consequently, the term "amino acid residue
difference relative to SEQ ID NO:2 at position Xn" as used herein
may refer to position "Xn" or to the corresponding position (e.g.
position (X-1)n) in a reference sequence that has been processed so
as to lack the starting methionine.
As used herein, the phrase "conservative amino acid substitutions"
refers to the interchangeability of residues having similar side
chains, and thus typically involves substitution of the amino acid
in the polypeptide with amino acids within the same or similar
defined class of amino acids. By way of example and not limitation,
in some embodiments, an amino acid with an aliphatic side chain is
substituted with another aliphatic amino acid (e.g., alanine,
valine, leucine, and isoleucine); an amino acid with a hydroxyl
side chain is substituted with another amino acid with a hydroxyl
side chain (e.g., serine and threonine); an amino acid having
aromatic side chains is substituted with another amino acid having
an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan,
and histidine); an amino acid with a basic side chain is
substituted with another amino acid with a basic side chain (e.g.,
lysine and arginine); an amino acid with an acidic side chain is
substituted with another amino acid with an acidic side chain
(e.g., aspartic acid or glutamic acid); and/or a hydrophobic or
hydrophilic amino acid is replaced with another hydrophobic or
hydrophilic amino acid, respectively. Exemplary conservative
substitutions are provided in Table 1.
TABLE-US-00001 TABLE 1 Exemplary Conservative Amino Acid
Substitutions Residue Potential Conservative Substitutions A, L, V,
I Other aliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M)
G, M Other non-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K,
R Other basic (K, R) N, Q, S, T Other polar H, Y, W, F Other
aromatic (H, Y, W, F) C, P Non-polar
As used herein, the phrase "non-conservative substitution" refers
to substitution of an amino acid in the polypeptide with an amino
acid with significantly differing side chain properties.
Non-conservative substitutions may use amino acids between, rather
than within, the defined groups and affects (a) the structure of
the peptide backbone in the area of the substitution (e.g., proline
for glycine) (b) the charge or hydrophobicity, or (c) the bulk of
the side chain. By way of example and not limitation, an exemplary
non-conservative substitution can be an acidic amino acid
substituted with a basic or aliphatic amino acid; an aromatic amino
acid substituted with a small amino acid; and a hydrophilic amino
acid substituted with a hydrophobic amino acid.
As used herein, "deletion" refers to modification of the
polypeptide by removal of one or more amino acids from the
reference polypeptide. Deletions can comprise removal of 1 or more
amino acids, 2 or more amino acids, 5 or more amino acids, 10 or
more amino acids, 15 or more amino acids, or 20 or more amino
acids, up to 10% of the total number of amino acids, or up to 20%
of the total number of amino acids making up the polypeptide while
retaining enzymatic activity and/or retaining the improved
properties of an engineered enzyme. Deletions can be directed to
the internal portions and/or terminal portions of the polypeptide.
In various embodiments, the deletion can comprise a continuous
segment or can be discontinuous.
As used herein, "insertion" refers to modification of the
polypeptide by addition of one or more amino acids to the reference
polypeptide. In some embodiments, the improved engineered
transglutaminase enzymes comprise insertions of one or more amino
acids to the naturally occurring transglutaminase polypeptide as
well as insertions of one or more amino acids to engineered
transglutaminase polypeptides. Insertions can be in the internal
portions of the polypeptide, or to the carboxy or amino terminus.
Insertions as used herein include fusion proteins as is known in
the art. The insertion can be a contiguous segment of amino acids
or separated by one or more of the amino acids in the naturally
occurring polypeptide.
The term "amino acid substitution set" or "substitution set" refers
to a group of amino acid substitutions in a polypeptide sequence,
as compared to a reference sequence. A substitution set can have 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid
substitutions. In some embodiments, a substitution set refers to
the set of amino acid substitutions that is present in any of the
variant transglutaminases listed in the Tables provided in the
Examples.
As used herein, "fragment" refers to a polypeptide that has an
amino-terminal and/or carboxy-terminal deletion, but where the
remaining amino acid sequence is identical to the corresponding
positions in the sequence. Fragments can typically have about 80%,
about 90%, about 95%0 about 98%, or about 99% of the full-length
transglutaminase polypeptide, for example the polypeptide of SEQ ID
NO:2. In some embodiments, the fragment is "biologically active"
(i.e., it exhibits the same enzymatic activity as the full-length
sequence).
As used herein, "isolated polypeptide" refers to a polypeptide that
is substantially separated from other contaminants that naturally
accompany it (e.g., proteins, lipids, and polynucleotides). The
term embraces polypeptides which have been removed or purified from
their naturally-occurring environment or expression system (e.g.,
host cell or in vitro synthesis). The improved transglutaminase
enzymes may be present within a cell, present in the cellular
medium, or prepared in various forms, such as lysates or isolated
preparations. As such, in some embodiments, the engineered
transglutaminase polypeptides of the present disclosure can be an
isolated polypeptide.
As used herein, "substantially pure polypeptide" refers to a
composition in which the polypeptide species is the predominant
species present (i.e., on a molar or weight basis it is more
abundant than any other individual macromolecular species in the
composition), and is generally a substantially purified composition
when the object species comprises at least about 50 percent of the
macromolecular species present by mole or % weight. Generally, a
substantially pure engineered transglutaminase polypeptide
composition comprises about 60% or more, about 70% or more, about
80% or more, about 90% or more, about 91% or more, about 92% or
more, about 93% or more, about 94% or more, about 95% or more,
about 96% or more, about 97% or more, about 98% or more, or about
99% of all macromolecular species by mole or % weight present in
the composition. Solvent species, small molecules (<500
Daltons), and elemental ion species are not considered
macromolecular species. In some embodiments, the isolated improved
transglutaminase polypeptide is a substantially pure polypeptide
composition.
As used herein, when used in reference to a nucleic acid or
polypeptide, the term "heterologous" refers to a sequence that is
not normally expressed and secreted by an organism (e.g., a
wild-type organism). In some embodiments, the term encompasses a
sequence that comprises two or more subsequences which are not
found in the same relationship to each other as normally found in
nature, or is recombinantly engineered so that its level of
expression, or physical relationship to other nucleic acids or
other molecules in a cell, or structure, is not normally found in
nature. For instance, a heterologous nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged in a manner not found in nature (e.g., a nucleic
acid open reading frame (ORF) of the invention operatively linked
to a promoter sequence inserted into an expression cassette, such
as a vector). In some embodiments, "heterologous polynucleotide"
refers to any polynucleotide that is introduced into a host cell by
laboratory techniques, and includes polynucleotides that are
removed from a host cell, subjected to laboratory manipulation, and
then reintroduced into a host cell.
As used herein, "suitable reaction conditions" refer to those
conditions in the biocatalytic reaction solution (e.g., ranges of
enzyme loading, substrate loading, cofactor loading, temperature,
pH, buffers, co-solvents, etc.) under which a transglutaminase
polypeptide of the present disclosure is capable of modifying a
substrate of interest. In some embodiments, the transglutaminases
cross-link substituted glutamines and substituted lysines in
various substrates, including low molecular weight substrates and
proteins. In some embodiments, the transglutaminases of the present
invention are capable of site specific modification of biological
macromolecules. Exemplary "suitable reaction conditions" are
provided in the present disclosure and illustrated by the
Examples.
As used herein, "loading," such as in "compound loading," "enzyme
loading," or "cofactor loading" refers to the concentration or
amount of a component in a reaction mixture at the start of the
reaction.
As used herein, "substrate" in the context of a biocatalyst
mediated process refers to the compound or molecule acted on by the
biocatalyst.
As used herein "product" in the context of a biocatalyst mediated
process refers to the compound or molecule resulting from the
action of the biocatalyst.
As used herein, "equilibration" as used herein refers to the
process resulting in a steady state concentration of chemical
species in a chemical or enzymatic reaction (e.g., interconversion
of two species A and B), including interconversion of
stereoisomers, as determined by the forward rate constant and the
reverse rate constant of the chemical or enzymatic reaction.
As used herein, "transglutaminase," "TG," "TGase," and "polypeptide
with transglutaminase activity," refer to an enzyme having the
ability to catalyze the acyl transfer reaction between the
gamma-carboxyamide group in a peptide/protein (e.g., glutamine
residues) and various primary amines, which act as amine donors. In
some embodiments, there is a substitution reaction of glutamine
with glutamic acid by the deamidation of glutamic acid. In some
embodiments, lysine is used as the acyl acceptor, which results in
the enrichment of the protein molecule used in the reaction. The
transfer of acyl onto a lysine residue in a polypeptide chain
induces the cross-linking process (i.e., the formation of intra- or
inter-molecular cross-links (See e.g., Kieliszek and Misiewicz,
supra, and Kashiwagi et al., J. Biol. Chem., 277:44252-44260
[2002]). In some embodiments, transglutaminases find use in
catalyzing deamination reactions in the absence of free amine
groups, but the presence of water, which acts as an acyl acceptor.
This results in significant changes in the physical and chemical
properties of affected proteins, including modifications in
viscosity, thermostability, elasticity, and resilience (See e.g.,
Kieliszek and Misiewicz, supra; Motoki and Seguroa, Trends Food
Sci. Technol., 9:204-210 [1998]; and Kuraishi et al., Food Rev.
Intl., 17:221-246 [2001]). Transglutaminases are known to be widely
distributed in various organisms, including humans, bacteria,
nematodes, yeasts, algae, plants, and lower vertebrates (See e.g.,
Santos and Tome, Recent Pat. Biotechnol., 3:166-174 [2009]).
As used herein, "transglutamination," "transamination," and
"transglutaminase reaction" refer to reactions in which the
gamma-glutaminyl of glutamine residue from a
protein/polypeptide/peptide is transferred to a primary amine or
the episilon-amino group of lysine or water, wherein an ammonia
molecule is released.
As used herein, "derived from" when used in the context of
engineered transglutaminase enzymes, identifies the originating
transglutaminase enzyme, and/or the gene encoding such
transglutaminase enzyme, upon which the engineering was based. For
example, the engineered transglutaminase enzyme of SEQ ID NO: 296
was obtained by artificially evolving, over multiple generations
the gene (SEQ ID NO: 1) encoding the S. mobaraensis
transglutaminase of SEQ ID NO:2.
Thus, this engineered transglutaminase enzyme is "derived from" the
naturally occurring or wild-type transglutaminase of SEQ ID NO:
2.
Transglutaminases
The present invention provides variant transglutaminases developed
from a wild-type S. mobaraensis transglutaminase enzyme. S.
mobaraensis is also classified as Streptoverticillium mobaraese.
This enzyme has a molecular weight of about 38 kDa and is calcium
independent (See e.g., Appl. Microbiol. Biotech., 64:447-454
[2004]: and US Pat. Appln. Publ. No. 2010/0099610, incorporated
herein by reference).
Transglutaminases have found use in altering the properties of
various peptides. In some embodiments, the enzyme is used to
cross-bind peptides useful in the food and dairy industries, as
well as in uses involving physiologically active peptides,
biomedicine, biomaterials, antibodies, the textile industry (e.g.,
wool and leather), methods for peptide conjugation, linkage of
agents to tissue, cosmetics, etc. (See e.g., EP 950 665. EP 785
276, WO 2005/070468, WO 2006/134148, WO 2008/102007, WO
2009/003732, U.S. Pat. No. 6,013,498, US Pat. Appln. Publ. No.
2010/0099610: US Pat. Appln. Publ. No. 2010/0249029; and US Pat.
Appln. Publ. No. 2010/0087371, each of which is incorporated by
reference herein; and Sato, Adv. Drug Deliv. Rev., 54:487-504
[2002]; Valdivia, J. Biotechnol., 122:326-333 [2006]; Wada,
Biotech. Lett., 23:1367-1372 [2001]; Kieliszek and Misiewicz, Folia
Microbiol., 59:241-250 [2014]; Yokoyama et al., Appl. Microbiol.
Biotechol., 64:447454 [2004]; Washizu et al., Biosci. Biotech.
Biochem., 58:82-87 [1994]; Kanaji et al., J. Biol. Chem.,
268:11565-11572 [1993]; Ando et al., Agric. Biol. Chem.
53:2613-2617 [1989]; Martins et al., Appl. Microbiol. Biotechnol.,
98:6957-6964 [2014]; Jerger et al., Angew. Chem. Int., 49:9995-9997
[2010]; Grunberg et al., PLoS ONE 8:e60350 [2013]: Mindt et al.,
Bioconj. Chem., 19:271-278 [2008]: Lhospice et al., Mol.
Pharmaceutics 12:1863-1871 [2015]; Dennler et al., Bioconj. Chem.,
25:569-578 [2014]; and Santos and Tome, Rec. Patents Biotechnol.,
3:166-174 [2009], for discussion of transglutaminases, their
sources, and uses). These enzymes are capable of improving the
firmness, viscosity, elasticity, and water-binding capacity of food
and other products.
In some embodiments, the transglutaminase variants provided herein
find use in the food industry for production of foods (e.g., jelly,
yogurt, cheese, noodles, chewing gum, candy, baked products,
soybean protein, gummy candy, snacks, pickles, meat, and
chocolate), while in some other embodiments, the transglutaminase
variants find use in other industries (e.g., textiles,
pharmaceuticals, diagnostics, etc.).
In some embodiments, the present invention provides engineered
transglutaminase polypeptides with amino acid sequences that have
at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO:
2, 6, 34, and/or 256.
In some embodiments, the engineered transglutaminase polypeptides
comprise substitutions at one or more positions selected from 79,
101, 101/201/212/287, 101/201/285, 101/287, and 327, wherein the
positions are numbered with reference to SEQ ID NO:6. In some
embodiments, the engineered transglutaminase polypeptides comprise
one or more substitutions selected from 79K, 101G,
101G/201K/212K/287G, 101G/201K/285Q, 101G/287G, and 327R, wherein
the positions are numbered with reference to SEQ ID NO:6. In some
embodiments, the engineered transglutaminase polypeptides comprise
one or more substitutions selected from S79K, Y101G,
Y101G/Q201K/R212K/S287G, Y101G/Q201K/R285Q, Y101G/S287G, and G327R,
wherein the positions are numbered with reference to SEQ ID
NO:6.
In some embodiments, the engineered transglutaminase polypeptides
comprise substitutions at one or more positions selected from 48,
48/67/70, 48/67/70/181/203/256, 48/67/70/181/256/345,
48/67/70/181/296/345/373, 48/67/70/203/256/296/345,
48/67/70/203/256/345/354/373, 48/67/70/203/345, 48/67/70/256,
48/67/70/256/296/345/373, 48/67/203/256/296/373, 48/67/203/256/345,
48/70/170/203, 48/70/203/254/296/343, 48/70/203/256/345/373,
48/70/203/256/345, 48/70/203/373, 48/170/203,
48/170/203/254/296/346, 48/170/203/254/296/346/373,
48/170/203/254/346/373, 48/170/203/254/346, 48/170/203/296/343/346,
48/170/203/296/346/373, 48/170/203/343/346, 48/170/203/346,
48/170/203/346/373, 48/170/203/373, 48/170/254, 48/170/296,
48/170/296/343/346, 48/170/343/346, 48/181, 48/181/203/256/345,
48/181/203/345, 48/181/256/296/345, 48/181/296, 48/181/296/345,
48/203, 48/203/254/296, 48/203/254/296/343/373,
48/203/254/296/346/373, 48/203/254/346, 48/203/254/346/373,
48/203/256, 48/203/256/296/345, 48/203/296/343/346/373,
48/203/296/343/373, 48/203/296/346, 48/203/296/346/373,
48/203/343/346, 48/203/343/346/373, 48/203/345, 48/203/346,
48/203/346/373, 48/254/296, 48/254/346, 48/256, 48/256/296,
48/256/296/345, 48/296/345, 48/296/373, 48/343/346, 48/345/373,
67/256, 67/296/345, 68/74/190/215/346, 68/136/215/255/282/297/346,
68/136/215/297/346, 68/136/234, 68/158/174/234/282/297/346,
68/158/215/297/346, 68/215/297/346, 68/234, 68/282/297/346,
68/297/346, 74/136/174/282/346, 74/136/174/297/346, 74/136/346,
74/158/255/297, 74/255/346, 74/346, 136/158/190/215/255/297/346,
136/158/215/297/346, 136/174/215/255/282/297/346,
136/190/215/297/346, 136/215/234/282/297, 136/215/234/297/346,
136/215/297, 136/297/346, 158/215/255/346, 158/215/346,
170/203/254/296/343/346, 170/203/254/343/373, 170/203/343/346,
174/190/234/297/346, 174/215/234/297/346, 174/215/255/297/346,
174/282/297/346, 190/255/282/346, 190/297/346, 203/296, 203/343,
203/343/346, 203/346, 215/255/297/346, 215/234/297/346,
215/255/297/346, 215/297, 215/297/346, 215/346, 234/255/346,
255/297/346, 255/346, 297/346, 343/346/373, and 346, wherein the
positions are numbered with reference to SEQ ID NO:2. In some
embodiments, the engineered transglutaminase polypeptides comprise
one or more substitutions selected from
48K/70L/203L/254Q/296L/343R, 48K/170K/203L, 48K/I
70K/203L/254Q/296L/346H, 48K/I 70K/203L/254Q/296L/346H/373M,
48K/170K/203L/254Q/346H/373M, 48K/170K/203L/254Q/346H,
48K/170K/203L/296L/343R/346H, 48K/170K/203L/296L/346H/373M,
48K/170K/203L/343R/346H, 48K/170K/203L/346H/373M,
48K/170K/203L/346H, 48K/170K/203L/373M, 48K/170K/254Q,
48K/170K/296L, 48K/170K/296L/343R/346H, 48K/170K/343R/346H,
48K/203L, 48K/203L/254Q/296L, 48K/203L/254Q/296L/343R/373M,
48K/203L/254Q/296L/346H/373M, 48K/203L/254Q/346H,
48K/203L/254Q/346H/373M, 48K/203L/296L/343R/346H/373M
48K/203L/296L/343R/373M, 48K/203L/296L/346H,
48K/203L/296L/346H/373M, 48K/203L/343R/346H,
48K/203L/343R/346H/373M, 48K/203L/346H, 48K/203L/346H/373M,
48K/254Q/346H, 48K/343R/346H, 48V, 48V/67E/70G,
48V/67E/70G/181K/203V/256G, 48V/67E/70G/181K/256G/345E,
48V/67E/70G/181K/296R/345E/373V, 48V/67E/70G/203V/256G/296R/345E,
48V/67E/70G/203V/345E, 48V/67E/70G/256G/296R/345E/373V,
48V/67E/70N/203V/256G/345E/354H/373L, 48V/67E/70N/256G,
48V/67E/203V/256G/296R/373V, 48V/67E/203V/256G/345E,
48K/70D/170K/203L, 48V/70G/203V/256G/345E/373V,
48V/70N/203V/256G/345E, 48V/70N/203V/373V, 48V/181K,
48V/181K/203V/256G/345E, 48V/181K/203V/345E,
48V/181K/256G/296R/345E, 48V/181K/296R, 48V/181K/296R/345E,
48V/203V, 48V/203V/256G, 48V/203V/256G/296R/345E, 48V/203V/345E,
48K/254Q/296L, 48V/256G, 48V/256G/296R, 48V/256G/296R/345E,
48V/296R/345E, 48V/296R/373V, 48V/345E/373L, 67E/256G,
67E/296R/345E, 68A/74T/190G/215N/346A,
68A/136Y/215N/255R/282K/297W/346A, 68A/136Y/215N/297W/346A,
68A/136Y/234Y, 68A/158I/174D/234Y/282K/297W/346A,
68A/158I/215N/297W/346A, 68A/215N/297W/346A, 68A/234Y,
68A/282K/297W/346A, 68A/297W/346A, 74T/136Y/174D/282K/346A,
74T/136Y/174D/297W/346A, 74T/136Y/346A, 74T/158I/255R/297W,
74T/255R/346A, 74T/346A, 136Y/158I/190G/215N/255R/297W/346A,
136Y/158I/215N/297W/346A, 136Y/174D/215N/255R/282K/297W/346A,
136Y/190G/215N/297W/346A, 136Y/215N/234Y/282K/297W,
136Y/215N/234Y/297W/346A, 136Y/215N/297W, 136Y/297W/346A,
158I/215N/255R/346A, 158I/215N/346A, 170K/203L/254Q/296L/343R/346H,
170K/203L/254Q/343R/373M, 170K/203L/343R/346H, I
74D/190G/234Y/297W/346A, 174D/215N/234Y/297W/346A,
174D/215N/255R/297W/346A, 174D/282K/297W/346A, 190G/255R/282K/346A,
190G/297W/346A, 203L/296L, 203L/343R/346H, 203L/343R, 203L/346H,
215H/255R/297W/346A, 215N/234Y/297W/346A, 215N/255R/297W/346A,
215N/297W, 215N/297W/346A, 215N/346A, 234Y/255R/346A,
255R/297W/346A, 255R/346A, 297W/346A, 343R/346H/373M, and 346A,
wherein the positions are numbered with reference to SEQ ID
NO:2.
In some embodiments, the engineered transglutaminase polypeptides
comprise one or more substitutions selected from
S48K/Y70L/G203L/R254Q/G296U/N343R, S48K/Q170K/G203L,
S48K/Q170K/G203L/R254Q/G296L/E346H,
S48K/Q170K/G203L/R254Q/G296L/E346H/K373M,
S48K/Q170K/G203L/R254Q/E346H/K373M, S48K/Q170K/G203U/R254Q/E346H,
S48K/Q170K/G203L/G296L/N343R/E346H,
S48K/Q170K/G203L/G296L/E346H/K373M, S48K/Q170K/G203L/N343R/E346H,
S48K/Q170K/G203L/E346H/K373M, S48K/Q170K/G203L/E346H,
S48K/Q170K/G203L/K373M, S48K/Q170K/R254Q, S48K/Q170K/G296L,
S48K/Q170K/G296L/N343R/E346H, S48K/Q170K/N343R/E346H, S48K/G203L,
S48K/G203L/R254Q/G296L, S48K/G203L/R254Q/G296L/N343R/K373M,
S48K/G203L/R254Q/G296L/E346H/K373M, S48K/G203L/R254Q/E346H,
S48K/G203L/R254Q/E346H/K373M, S48K/G203L/G296L/N343R/E346H/K373M,
S48K/G203L/G296L/N343R/K373M, S48K/G203L/G296L/E346H,
S48K/G203L/G296L/E346H/K373M, S48K/G203L/N343R/E346H,
S48K/G203L/N343R/E346H/K373M, S48K/G203L/E346H,
S48K/G203L/E346H/K373M, S48K/R254Q/E346H, S48K/N343R/E346H, S48V,
S48V/R67E/Y70G, S48V/R67E/Y70G/R181K/G203V/S256G,
S48V/R67E/Y70G/R181K/S256G/S345E,
S48V/R67E/Y70G/R181K/G296R/S345E/K373V,
S48V/R67E/Y70G/G203V/S256G/G296R/S345E, S48V/R67E/Y70G/G203V/S345E,
S48V/R67E/Y70G/S256G/G296R/S345E/K373V,
S48V/R67E/Y70N/G203V/S256/S345E/G354H/K373L, S48V/R67E/Y70N/S256G,
S48V/R67E/G203V/S256G/G296R/K373V, S48V/R67E/G203V/S256G/S345E,
S48K/Y70D/Q170K/G203L, S48V/Y70G/G203V/S256G/S345E/K373V,
S48V/Y70N/G203V/S256G/S345E, S48V/Y70N/G203V/K373V, S48V/R181K,
S48V/R181K/G203V/S256G/S345E, S48V/R181K/G203V/S345E,
S48V/R181K/S256G/G296R/S345E, S48V/R181G296R,
S48V/R181K/G296R/S345E, S48V/G203V, S48V/G203V/S256G,
S48V/G203V/S256G/G296R/S345E, S48V/G203V/S345E, S48K/R254Q/G296L,
S48V/S256G, S48V/S256G/G296R, S48V/S256G/G296R/S345E,
S48V/G296R/S345E, S48V/G296R/K373V, S48V/S345E/K373L, R67E/S256G,
R67E/G296R/S345E, P68A/E74T/S190G/P215N/E346A,
P68A/F136Y/P215N/S255R/R282K/F297W/E346A,
P68A/F136Y/P215N/F297W/E346A, P68A/F136Y/H234Y,
P68A/V158I/E174D/H234Y/R282K/F297W/E346A,
P68A/V158I/P215N/F297W/E346A, P68A/P215N/F297W/E346A, P68A/H234Y,
P68A/R282K/F297W/E346A, P68A/F297W/E346A,
E74T/F136Y/E174D/R282K/E346A, E74T/F136Y/E174D/F297W/E346A,
E74T/F136Y/E346A, E74T/V158I/S255R/F297W, E74T/S255R/E346A,
E74T/E346A, F136Y/V158I/S190G/P215N/S255R/F297W/E346A,
F136Y/V158I/P215N/F297W/E346A,
F136Y/E174D/P215N/S255R/R282K/F297W/E346A,
F136Y/S190G/P215N/F297W/E346A, F136Y/P215N/H234Y/R282K/F297W,
F136Y/P215N/H234Y/F297W/E346A, F136Y/P215N/F297W,
F136Y/F297W/E346A, V158I/P215N/S255R/E346A, V158I/P215N/E346A,
Q170K/G203L/R254Q/G296L/N343R/E346H, Q170K/G203L/R254Q/N343R/K373M,
Q170K/G203L/N343R/E346H, E174D/S190G/H234Y/F297W/E346A,
E174D/P215N/H234Y/F297W/E346A, E174D/P215N/S255R/F297W/E346A,
E174D/R282K/F297W/E346A, S190G/S255R/R282K/E346A,
S190G/F297W/E346A, G203L/G296L, G203L/N343R/E346H, G203L/N343R,
G203L/E346H, P215H/S255R/F297W/E346A, P215N/H234Y/F297W/E346A,
P215N/S255R/F297W/E346A, P215N/F297W, P215N/F297W/E346A,
P215N/E346A, H234Y/S255R/E346A, S255R/F297W/E346A, S255R/E346A,
F297W/E346A, N343R/E346H/K373M, and E346A, wherein the positions
are numbered with reference to SEQ ID NO:2.
In some embodiments, the engineered transglutaminase polypeptides
comprise substitutions at one or more positions selected from
33/67/70/181/203/256/296/373, 36/48/203/254/346,
48/67/70/181/203/256/296/373, 48/67/70/203/256/296/373,
48/67/181/203/256/296/373, 48/67/181/203/256/373,
48/67/181/256/296, 48/67/203/256/296/373/378, 48/67/203/256/373,
48/67/203/296/373, 48/67/256/296/373, 48/70/181/203/256/296/373,
48/70/181/203/256/373, 48/70/181/203/296/373,
48/70/203/256/296/373, 48/70/203/256/373, 48/70/203/296,
48/70/203/296/373, 48/70/203/373, 48/70/256/296/373, 48/70/296/373,
48/176/203/254/346/373, 48/181/203/256/296/373, 48/181/203/256/373,
48/181/203/296, 48/181/203/373, 48/181/256/296/373, 48/203/254,
48/203/254/343, 48/203/254/343/346/373, 48/203/254/343/355/373,
48/203/254/343/373, 48/203/254/346/373, 48/203/254/373,
48/203/256/296, 48/203/256/296/373, 48/203/256/373, 48/203/296/373,
48/203/296/373/374, 48/203/343/373, 48/203/373, 48/254,
48/254/343/346/373, 48/254/343/373, 48/254/346/373, 48/254/373,
48/256/296/373, 48/256/373, 48/373, 67/70/181/203/256/296/373,
67/70/181/256/296/373, 67/70/181/373, 67/181/203/256/296,
67/181/203/256/296/373, 67/181/203/256/373, 67/203/256/296/373,
67/256/296/373, 70/181/203/256/296/373, 70/181/203/296/373, 70/203,
70/203/256/296/373, 70/203/256/373, 70/203/296/373,
74/136/215/234/282/297/346, 74/136/215/234/282/346,
74/136/215/234/297, 74/136/215/234/297/343/346,
74/136/215/234/297/346, 74/136/215/234/346, 74/136/215/282/297/346,
74/136/215/282/346, 74/136/215/297/346, 74/136/215/346,
74/136/234/282/297/346, 74/136/234/346, 74/136/282/297/346, 74/215,
74/215/234/282/297/346, 74/215/282/297/346, 74/215/346,
136/215/234/282/297/346, 136/215/282/297, 136/215/282/297/346,
136/215/282/346, 136/215/297/346, 136/215/346, 136/234/297,
136/234/297/346, 136/234/346, 136/282/297, 181/203/256,
181/203/256/296, 181/203/256/296/373, 181/203/256/373,
181/203/296/373, 181/203/373, 181/256/296/373, 181/296,
203/224/254/373, 203/254, 203/254/343/346/373, 203/254/343/373,
203/254/346, 203/254/346/373, 203/254/373, 203/346/373, 203/373,
203/209/256/373, 203/256, 203/256/296, 203/256/296/320/373,
203/256/296/373, 203/256/296/373/386, 203/256/373, 203/296/373,
203/373, 215/234/282/297/346, 215/234/282/346, 215/234/346,
234/282/346, 254, 254/346, 254/346/373, 254/373, 256/296,
256/296/373, 256/373, 282/297/346, 343/373, and 373, wherein the
positions are numbered with reference to SEQ ID NO:2.
In some embodiments, the engineered transglutaminase polypeptides
comprise one or more substitutions selected from
33D/67E/70G/181K/203V/256G/296R/373V, 36E/48K/203L/254Q/346H,
48K/176T/203L/254Q/346H/373M, 48K/203L/254Q, 48K/203L/254Q/343R,
48K/203L/254Q/343R/346H/373M, 48K/203L/254Q/343R/355T/373M,
48K/203L/254Q/343R/373M, 48K/203L/254Q/346D/373M,
48K/203L/254Q/373M, 48K/203L/343R/373M, 48K/203L/373M, 48K/254Q,
48K/254Q/343R/346H/373M, 48K/254Q/343R/373M, 48K/254Q/346H/373M,
48K/254Q/373M, 48V/67E/70G/181K/203V/256G/296R/373V,
48V/67E/70G/203V/256G/296R/373V, 48V/67E/181K/203V/256G/296R/373V,
48V/67E/181K/203V/256G/373V, 48V/67E/181K/256G/296R,
48V/67E/203V/256G/296R/373V/378D, 48V/67E/203V/256G/373V,
48V/67E/203V/296R/373V, 48V/67E/256G/296R/373V,
48V/70G/181K/203V/256G/296R/373V, 48V/70G/181K/203V/256G/373V,
48V/70G/181K/203V/296R/373V, 48V/70G/203V/256G/296R/373V,
48V/70G/203V/256G/373V, 48V/70G/203V/296R, 48V/70G/203V/296R/373V,
48V/70G/203V/373V, 48V/70G/256G/296R/373V, 48V/70G/296R/373V,
48V/181K/203V/256G/296R/373V, 48V/181K/203V/256G/373V,
48V/181K/203V/296R, 48V/181K/203V/373V, 48V/181K/256G/296R/373V,
48V/203V/256G/296R, 48V/203V/256G/296R/373V, 48V/203V/256G/373V,
48V/203V/296R/373V, 48V/203V/296R/373V/374L, 48V/203V/373V,
48V/256G/296R/373V, 48V/256G/373V, 48V/373V,
67E/70G/181K/203V/256G/296R/373V, 67E/70G/181K/256G/296R/373V,
67E/70G/181K/373V, 67E/181K/203V/256G/296R,
67E/181K/203V/256G/296R/373V, 67E/181K/203V/256G/373V,
67E/203V/256G/296R/373V, 67E/256G/296R/373V,
70G/181K/203V/256G/296R/373V, 70G/181K1203V/296R/373V, 70G/203V,
70G/203V/256G/296R/373V, 70G/203V/256G/373V, 70G/203V/296R/373V,
74T/136Y/215N/234Y/282K/297W/346A, 74T/136Y/215N/234Y/282K/346A,
74T/136Y/215N/234Y/297W, 74T/136Y/215N/234Y/297W/343Y/346A,
74T/136Y/215N/234Y/297W/346A, 74T/136Y/215N/234Y/346A,
74T/136Y/215N/282K/297W/346A, 74T/136Y/215N/282K/346A,
74T/136Y/215N/297W/346A, 74T/136Y/215N/346A,
74T/136Y/234Y/282K/297V/346A, 74T/136Y/234Y/346A,
74T/136Y/282K/297W/346A, 74T/215N, 74T/215N/234Y/282K/297W/346A,
74T/215N/282K/297W/346A, 74T/215N/346A,
136Y/215N/234Y/282K/297W/346A, 136Y/215N/282K/297W,
136Y/215N/282K/297W/346A, 136Y/215N/282K/346A, 136Y/215N/297W/346A,
136Y/215N/346A, 136Y/234Y/297W, 136Y/234Y/297W/346A,
136Y/234Y/346A, 136Y/282K/297W, 181K/203V/256G,
181K/203V/256G/296R, 181K/203V/2566/296R/373V, 181K1203V/256G/373V,
181K/203V/296R/373V, 181K/203V/373V, 181K/256G/296R/373V,
181K/296R, 203L/224T/254Q/373M, 203L/254Q,
203L/254Q/343R/346H/373M, 203L/254Q/343R/373M, 203L/254Q/346H,
203L/254Q/346H/373M, 203L/254Q/373M, 203L/346H/373M, 203L/373M,
203V/209Y/256G/373V, 203V/256G, 203V/256G/296R,
203V/256G/296R/320Y/373V, 203V/256G/296R/373V,
203V/256G/296R/373V/386Y, 203V/256G/373V, 203V/296R/373V,
203V/373V, 215N/234Y/282K/297W/346A, 215N/234Y/282K/346A,
215N1234Y/346A, 234Y/282K/346A, 254Q, 254Q/346H, 254Q/346H/373M,
254Q/373M, 256G/296R, 256G/296R/373V, 256G/373V, 282K/297W/346A,
343R/373M, and 373M/V, wherein the positions are numbered with
reference to SEQ ID NO:2. In some embodiments, the engineered
transglutaminase polypeptides comprise one or more substitutions
selected from A33D/R67E/Y70G/R181K/G2.beta.V/S256G/G296R/K373V,
A36E/S48K/G203L/R254Q/E346H, S48K/A176T/G203L/R254Q/E346H/K373M,
S48K/G203U/R254Q, S48K/G203L/R254Q/N343R,
S48K/G203L/R254Q/N343R/E346H/K373M,
S48K/G203L/R254Q/N343R/A355T/K373M, S48K/G203L/R254Q/N343R/K373M,
S48K/G203L/R254Q/E346D/K373M, S48K/G203L/R254Q/K373M,
S48K/G203L/N343R/K373M, S48K/G203L/K373M, S48K/R254Q,
S48K/R254Q/N343R/E346H/K373M, S48K/R254Q/N343R/K373M,
S48K/R254Q/E346H/K373M, S48K/R254Q/K373M,
S48V/R67E/Y70G/R181K/G203V/S256G/G296R/K373V,
S48V/R67E/Y70G/G203V/S256G/G296R/K373V,
S48V/R67E/R181K/G203V/S256G/G296R/K373V,
S48V/R67E/R181K/G203V/S256G/K373V, S48V/R67E/R181K/S256G/G296R,
S48V/R67E/G203V/S256G/G296R/K373V/G378D,
S48V/R67E/G203V/S256G/K373V, S48V/R67E/G203V/G296R/K373V,
S48V/R67E/S256G/G296R/K373V,
S48V/Y70G/R181K/G203V/S256G/G296R/K373V,
S48V/Y70G/R181K/G203V/S256G/K373V,
S48V/Y70G/R181K/G203V/G296R/K373V,
S48V/Y70G/G203V/S256/G296R/K373V, S48V/Y70G/G203V/S256G/K373V,
S48V/Y70G/G203V/G296R, S48V/Y70/G203V/G296R/K373V,
S48V/Y70G/G203V/K373V, S48V/Y70/S256G/G296R/K373V,
S48V/Y70G/G296R/K373V, S48V/R181K/G203V/S256G/G296R/K373V,
S48V/R181K/G203V/S256G/K373V, S48V/R181K/G203V/G296R,
S48V/R181K/G203V/K373V, S48V/R181K/S256G/G296R/K373V,
S48V/G203V/S256G/G296R, S48V/G203V/S256/G296R/K373V,
S48V/G203V/S256G/K373V, S48V/G203V/G296R/K373V,
S48V/G203V/G296R/K373V/Q374L, S48V/G203V/K373V,
S48V/S256G/G296R/K373V, S48V/S256G/K373V, S48V/K373V,
R67E/Y70G/R181K/G203V/S256G/G296R/K373V,
R67E/Y70G/R181K/S256G/G296R/K373V, R67E/Y70G/R181K/K373V,
R67E/R181K/G203V/S256G/G296R, R67E/R181K/G203V/S256G/G296R/K373V,
R67E/R181K/G203V/S256G/K373V, R67E/G203V/S256G/G296R/K373V,
R67E/S256/G296R/K373V, Y70G/R181K/G203V/S256G/G296R/K373V,
Y70G/R181K/G203V/G296R/K373V, Y70G/G203V,
Y70G/G203V/S256/G296R/K373V, Y70G/G203V/S256G/K373V,
Y70G/G203V/G296R/K373V, E74T/F136Y/P215N/H234Y/R282K/F297W/E346A,
E74T/F136Y/P215N/H234Y/R282K/E346A, E74T/F136Y/P215N/H234Y/F297W,
E74T/F136Y/P215N/H234Y/F297W/N343Y/E346A,
E74T/F136Y/P215N/H234Y/F297W/E346A, E74T/F136Y/P215N/H234Y/E346A,
E74T/F136Y/P215N/R282K/F297W/E346A, E74T/F136Y/P215N/R282K/E346A,
E74T/F136Y/P215N/F297W/E346A, E74T/F136Y/P215N/E346A,
E74T/F136Y/H234Y/R282K/F297W/E346A, E74T/F136Y/H234Y/E346A,
E74T/F136Y/R282K/F297W/E346A, E74T/P215N,
E74T/P215N/H234Y/R282K/F297W/E346A, E74T/P215N/R282K/F297W/E346A,
E74T/P215N/E346A, F136Y/P215N/H234Y/R282K/F297W/E346A,
F136Y/P215N/R282K/F297W, F136Y/P215N/R282K/F297W/E346A,
F136Y/P215N/R282K/E346A, F136Y/P215N/F297W/E346A,
F136Y/P215N/E346A, F136Y/H234Y/F297W, F136Y/H234Y/F297W/E346A,
F136Y/H234Y/E346A, F136Y/R282K/F297W, R181K/G203V/S256G,
R181K/G203V/S256G/G296R, R181K/G203V/S256G/G296R/K373V,
R181K/G203V/S256G/K373V, R181K/G203V/G296R/K373V,
R181K/G203V/K373V, R181K/S256G/G296R/K373V, R181K/G296R,
G203L/P224T/R254Q/K373M, G203L/R254Q,
G203L/R254Q/N343R/E346H/K373M, G203L/R254Q/N343R/K373M,
G203L/R254Q/E346H, G203L/R254Q/E346H/K373M, G203U/R254Q/K373M,
G203L/E346H/K373M, G203U/K373M, G203V/N209Y/S256G/K373V,
G203V/S256G, G203V/S256G/G296R, G203V/S256/G296R/H320Y/K373V,
G203V/S256G/G296R/K373V, G203V/S256/G296R/K373V/H386Y,
G203V/S256G/K373V, G203V/G296R/K373V, G203V/K373V,
P215N/H234Y/R282K/F297W/E346A, P215N/H234Y/R282K/E346A,
P215N/H234Y/E346A, H234Y/R282K/E346A, R254Q, R254Q/E346H,
R254Q/E346H/K373M, R254Q/K373M, S256G/G296R, S256G/G296R/K373V,
S256G/K373V, R282K/F297W/E346A, N343R/K373M, and K373M/V, wherein
the positions are numbered with reference to SEQ ID NO:2.
In some embodiments, the engineered transglutaminase polypeptides
comprise substitutions at one or more positions selected from
48/49, 49, 50, 50, 331, 291, 292, 330, and 331, wherein the
positions are numbered with reference to SEQ ID NO:34. In some
embodiments, the engineered transglutaminase polypeptides comprise
one or more substitutions selected from 48S/49W, 49Y, 50A/F/Q/R,
331H/P/V, 291C, 292R. 330H/Y, and 331R, wherein the positions are
numbered with reference to SEQ ID NO:34. In some embodiments, the
engineered transglutaminase polypeptides comprise one or more
substitutions selected from K48S/D49W, D49Y, D50A/F/Q/R, L331H/P/V,
T291C, S292R, S330H/Y, and L331R, wherein the positions are
numbered with reference to SEQ ID NO:34.
In some embodiments, the engineered transglutaminase polypeptides
comprise substitutions at one or more positions selected from
27/48/67/70/74/234/256/282/346/373,
27/48/67/70/136/203/215/256/282/346/373, 27/48/67/70/346/373,
27/48/67/74/203/256/346/373, 27/67/234/296/373, 45/287/328/333,
45/292/328, 48, 48/284/292/333, 48/287/292/297, 48/287/297/328/333,
48/292, 48/292/297, 48/49/50/292/331, 48/49/50/292, 48/49/50/331,
48/49/330/331, 48/49/50/349, 48/49/50/291/292/331,
48/49/50/292/331, 48/67/70/203/215/234/256/346,
48/67/70/234/256/282/297/346, 48/67/70/346,
48/67/74/203/234/256/282/346/373, 48/67/74/234/297/346/373,
48/67/74/346, 48/67/203/346/373, 48/67/234/256/297/346/373,
48/67/234/256/346/373, 48/67/215/282/297/346/373, 48/67/346/373,
48/70/74/297/346/373, 48/70/203/215/256/282/346/373,
48/70/215/234/256/346/373, 48/74/203/234/256/346/373,
48/74/234/256/297/346/373, 48/136/256/346/373,
48/203/234/256/297/346/373, 48/203/234/256/346/373,
48/203/234/346/373, 48/203/296/373, 48/215/234/346/373,
48/215/346/373, 48/234/256/296/346/373, 48/234/256/346/373,
48/256/373, 49/50/292/331, 49/50/292/331/349, 49/50/331,
49/50/331/349, 50, 67/70/74/136/203/215/256/346/373,
67/70/74/203/215/234/346/373, 67/70/74/215/234/297/346/373,
67/70/74/215/256/373, 67/70/136/203/297/346/373,
67/70/203/215/256/346/373, 67/70/203/373, 67/70/215, 67/74/136,
67/74/203/234/256, 67/74/215/256/297/346/373, 67/74/215/346/373,
67/74/256/346/373, 67/136/203/215/256/346/373,
67/136/203/256/346/373, 67/203/234/256/346/373, 67/203/297/346/373,
67/215/234/297/346/373, 67/297/346, 70/74/203/215/346/373, 136,
136/346/373, 203/234/346, 203/234/346/373, 203/373, 234/282, 287,
234/346/373, 287/292, 287/292/295/297, 287/292/297, 287/295/297,
287/330/333, 292, 292/297, 292/330/331, 292/330/331, 292/331,
292/331/349, 292/349, 295, 295/297/333, 297/328, 297/373, 328/333,
330, 330/331, 331, 331/349, 333, 346/373, and 373, wherein the
positions are numbered with reference to SEQ ID NO:256. In some
embodiments, the engineered transglutaminase polypeptides comprise
one or more substitutions selected from
27S/48V/67E170G/74T/234Y/256G/282K/346A/373L,
27S/48V/67E/70G/136Y/203V/215H/256G/282K/346A/373V,
27S/48V/67E/70G/346A/373L, 27S/48V/67E/74T/203V/256G/346A/373M,
27S/67E/234Y/296R/373M, 45S/287S/328E/333P, 45S/292K/328E, 48A,
48A/284G/292K/333P, 48A/287S/292K/297Y, 48A/287S/297Y/328E/333P,
48A/292K, 48A/292K1297Y, 48S/49G/50A/292R/331P, 48S/49W/50A/292R,
48S/49W/50A/331V, 48S/491W/330Y/331V, 48S/49W/50A/349R,
48S/49Y/50A/291C/292R/331V, 48 S/49Y/50Q/292R/331V,
48V/67E/70G/203V/215H/234Y/256G/346A,
48V/67E/70G/234Y/256/282K/297W/346A, 48V/67E/70G/346A,
48V/67E174T/203V/234Y/256G/282K/346A/373M,
48V/67E/74T/234Y/297W/346A/373M, 48V/67E/74T/346A,
48V/67E/203V/346A/373M, 48V/67E/215H/282K/297W/346A/373M,
48V/67E/234Y/256G/297W/346A/373V, 48V/67E/234Y/256G/346A/373M,
48V/67E/346A/373M, 48V/70G/74T/297W/346A/373M,
48V/70G/203V/215H/256G/282K/346A/373V,
48V/70G/215H/234Y/256G/346A/373M, 48V/74T/203V/234Y/256G/346A/373V,
48V/74T/234Y/256/297W/346A/373V, 48V/136Y/256G/346A/373M,
48V/203V/234Y/256G/297W/346A/373V, 48V/203V/234Y/256G/346A/373M,
48V/203V/234Y/346A/373M, 48V/203V/296R/373M,
48V/215H/234Y/346A/373V, 48V/215H/346A/373M,
48V/234Y/256G/296R/346A/373M, 48V/234Y/256/346A/373M,
48V/256G/373L, 49/50A/292R/331V, 49G/50Q/292R/331V/349R,
49W/50A/331V, 49W/50A/331V/349R, 50A,
67E/70G/74T/136Y/203V/215H/256G/346A/373M,
67E/70G/74T/203V/215H/234Y/346A/373V,
67E/70G/74T/215H/234Y/297W/346A/373L, 67E/70G/74T/215H/256G/373M,
67E/70G/136Y/203V/297W/346A/373M, 67E/70G/203V/215H/256G/346A/373L,
67E/70G/203V/373M, 67E/70G/215H, 67E/74T/136Y,
67E/74T/203V/234Y/256G, 67E/74T/215H/256G/297W/346A/373L,
67E/74T/215H/346A/373V, 67E/74T/256G/346A/373M,
67E/136Y/203V/215H/256G/346A/373V, 67E136Y/203V/256G/346A/373M,
67E/203V/234Y/256G/346A/373V, 67E/203V/297W/346A/373M,
67E/215H/234Y/297W/346A/373V, 67E/297W/346A,
70G/74T/203V/215H/346A/373V, 136Y, 136Y/346A/373M, 203V/234Y/346A,
203V/234Y/346A/373V, 203V/373M, 234Y/282K, 234Y/346A/373M, 287S,
287S/292K, 287S/292K/295R/297Y, 287S/292K/297Y, 287S/295R/297Y,
287S/330G/333P, 292K, 292K1297Y, 292R, 292R/330Y/331P,
292R/330Y/331V, 292R/331V, 292R/331V/349R, 292134911, 295R,
295R/297Y/333P, 297Y/328E, 297W/373M, 328E/333P, 330Y, 330Y/331P,
331V, 331V/349R, 333P, 346A/373V, and 373M/V, wherein the positions
are numbered with reference to SEQ ID NO:256, In some embodiments,
the engineered transglutaminase polypeptides comprise one or more
substitutions selected from
N27S/K48V/R67E/Y70G/E74T/H234Y/S256G/R282K/H346A/K373L,
N27S/K48V/R67E/Y70G/F136Y/L203V/P215H/S256G/R282K/H346A/K373V,
N27S/K48V/R67E/Y70G/H346A/K373L,
N27S/K48V/R67E/E74T/L203V/S256/H346A/K373M,
N27S/R67E/H234Y/G296R/K373M, A45S/P287S/N328E/A333P,
A45S/S292K/N328E, K48A, K48A/R284G/S292K/A333P,
K48A/P287S/S292K/F297Y, K48A/P287S/F297Y/N328E/A333P, K48A/S292K,
K48A/S292K/F297Y, K48S/D49G/R50A/S292R/L331P, K48S/D49W/R50A/S292R,
K48S/D49W/R50A/L331V, K48S/D49W/S330Y/L331V, K48S/D49W/R50A/S349R,
K48S/D49Y/R50A/T291C/S292R/L331V, K48S/D49Y/R50Q/S292R/L331V,
K48V/R67E/Y70G/L203V/P215H/H234Y/S256G/H346A,
K48V/R67E/Y70G/H234Y/S256G/R282K/F297W/H346A, K48V/R67E/Y70G/H346A,
K48V/R67E/E74T/L203V/H234Y/S256/R282K/H346A/K373M,
K48V/R67E/E74T/H234Y/F297W/H346A/K373M, K48V/R67E/E74T/H346A,
K48V/R67E/L203V/H346A/K373M,
K48V/R67E/P215H/R282K/F297W/H346A/K373M,
K48V/R67E/H234Y/S256G/F297W/H346A/K373V,
K48V/R67E/H234Y/S256G/H346A/K373M, K48V/R67E/H346A/K373M,
K48V/Y70G/E74T/F297W/H346A/K373M,
K48V/Y70G/L203V/P215H/S256G/R282K/H346A/K373V,
K48V/Y70G/P215H/H234Y/S256G/H346A/K373M,
K48V/E74T/L203V/H234Y/S256G/H346A/K373V,
K48V/E74T/H234Y/S256G/F297W/H346A/K373V,
K48V/F136Y/S256/H346A/K373M,
K48V/L203V/H234Y/S256/F297W/H346A/K373V,
K48V/L203V/H234Y/S256G/H346A/K373M, K48V/L203V/H234Y/H346A/K373M,
K48V/L203V/G296R/K373M, K48V/P215H/H234Y/H346A/K373V,
K48V/P215H/H346A/K373M, K48V/H234Y/S256/G296R/H346A/K373M,
K48V/H234Y/S256/H346A/K373M, K48V/S256G/K373L,
D49G/R50A/S292R/L331V, D49G/R50Q/S292R/L331V/S349R,
D49W/R50A/L331V, D49W/R50A/L331V/S349R, R50A,
R67E/Y70G/E74T/F136Y/L203V/P215H/S256G/H346A/K373M,
R67E/Y70G/E74T/L203V/P215H/H234Y/H346A/K373V,
R67E/Y700/E74T/P215H/H234Y/F297W/H346A/K373L,
R67E/Y70G/E74T/P215H/S256G/K373M,
R67E/Y70G/F136Y/L203V/F297W/H346A/K373M,
R67E/Y70G/L203V/P215H/S256G/H346A/K373L, R67E/Y70G/L203V/K373M,
R67E/Y70G/P215H, R67E/E74T/F136Y, R67E/E74T/L203V/H234Y/S256G,
R67E/E74T/P215H/S2560/F297W/H346A/K373L,
R67E/E74T/P215H/H346A/K373V, R67E/E74T/S256G/H346A/K373M,
R67E/F136Y/L203V/P215H/S256G/H346A/K373V,
R67E/F136Y/L203V/S256G/H346A/K373M,
R67E/L203V/H234Y/S256G/H346A/K373V, R67E/L203V/F297W/H346A/K373M,
R67E/P215H/H234Y/F297W/H346A/K373V, R67E/F297W/H346A,
Y70G/E74T/L203V/P215H/H346A/K373V, F136Y, F136Y/H346A/K373M,
L203V/H234Y/H346A, L203V/H234Y/H346A/K373V, L203V/K373M,
H234Y/R282K, H234Y/H346A/K373M, P287S, P287S/S292K,
P287S/S292K/E295R/F297Y, P287S/S292K/F297Y, P287S/E295R/F297Y,
P287S/S330G/A333P, S292K, S292K/F297Y, S292R, S292R/S330Y/L331P,
S292R/S330Y/L331V, S292R/L33IV, S292R/L331V/S349R, S292R/S349R,
E295R, E295R/F297Y/A333P, F297Y/N328E, F297W/K373M, N328E/A333P,
S330Y, S330Y/L331P, L331V, L331V/S349R, A333P, H346A/K373V, and
K373M/V, wherein the positions are numbered with reference to SEQ
ID NO:256.
The present invention also provides polynucleotides encoding the
engineered transglutaminase polypeptides. In some embodiments, the
polynucleotides are operatively linked to one or more heterologous
regulatory sequences that control gene expression, to create a
recombinant polynucleotide capable of expressing the polypeptide.
Expression constructs containing a heterologous polynucleotide
encoding the engineered transglutaminase polypeptides can be
introduced into appropriate host cells to express the corresponding
transglutaminase polypeptide.
Because of the knowledge of the codons corresponding to the various
amino acids, availability of a protein sequence provides a
description of all the polynucleotides capable of encoding the
subject. The degeneracy of the genetic code, where the same amino
acids are encoded by alternative or synonymous codons allows an
extremely large number of nucleic acids to be made, all of which
encode the improved transglutaminase enzymes disclosed herein.
Thus, having identified a particular amino acid sequence, those
skilled in the art could make any number of different nucleic acids
by simply modifying the sequence of one or more codons in a way
which does not change the amino acid sequence of the protein. In
this regard, the present disclosure specifically contemplates each
and every possible variation of polynucleotides that could be made
by selecting combinations based on the possible codon choices, and
all such variations are to be considered specifically disclosed for
any polypeptide disclosed herein, including the amino acid
sequences presented in the Tables in the Examples herein.
In various embodiments, the codons are preferably selected to fit
the host cell in which the protein is being produced. For example,
preferred codons used in bacteria are used to express the gene in
bacteria: preferred codons used in yeast are used for expression in
yeast; and preferred codons used in mammals are used for expression
in mammalian cells.
In some embodiments, all codons need not be replaced to optimize
the codon usage of the transglutaminase polypeptides since the
natural sequence will comprise preferred codons and because use of
preferred codons may not be required for all amino acid residues.
Consequently, codon optimized polynucleotides encoding the
transglutaminase enzymes may contain preferred codons at about 40%,
50%, 60%, 70%, 80%, or greater than 90% of codon positions of the
full length coding region.
In some embodiments, the polynucleotide comprises a nucleotide
sequence encoding a transglutaminase polypeptide with an amino acid
sequence that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity
to SEQ ID NO: 2, 6, 34, and/or 256. In some embodiments, the
polynucleotide comprises a nucleotide sequence encoding a
transglutaminase polypeptide with an amino acid sequence that has
at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%/0, or 99% or more sequence identity to SEQ ID NO: 2,
6, 34, and/or 256. In some embodiments, the polynucleotide encodes
a transglutaminase amino acid sequence of SEQ ID NO: 2, 6, 34,
and/or 256. In some embodiments, the present invention provides
polynucleotide sequences having at least about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%/0, 98%, or 99% or more
sequence identity to SEQ ID NO: 1, 5, 33, and/or 255. In some
embodiments, the present invention provides polynucleotide
sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to
SEQ ID NO: 1, 5, 33, and/or 255.
In some embodiments, the isolated polynucleotide encoding an
improved In some embodiments, the polynucleotide comprises a
nucleotide sequence encoding a transglutaminase polypeptide with an
amino acid sequence that has at least about 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more
sequence identity to SEQ ID NO: 2, 6, 34, and/or 256. The
polypeptide is manipulated in a variety of ways to provide for
improved activity and/or expression of the polypeptide.
Manipulation of the isolated polynucleotide prior to its insertion
into a vector may be desirable or necessary depending on the
expression vector. The techniques for modifying polynucleotides and
nucleic acid sequences utilizing recombinant DNA methods are well
known in the art.
For example, mutagenesis and directed evolution methods can be
readily applied to polynucleotides to generate variant libraries
that can be expressed, screened, and assayed.
Mutagenesis and directed evolution methods are well known in the
art (See e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721,
5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970,
6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861,
6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713,
6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356,
6,352,859, 6,355,484, 6,358,740, 6,358,742, 6,365,377, 6,365,408,
6,368,861, 6,372,497, 6,337,186, 6,376,246, 6,379,964, 6,387,702,
6,391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745,
6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468,
6,455,253, 6,479,652, 6,482,647, 6,483,011, 6,484,105, 6,489,146,
6,500,617, 6,500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065,
6,521,453, 6,528,311, 6,537,746, 6,573,098, 6,576,467, 6,579,678,
6,586,182, 6,602,986, 6,605,430, 6,613,514, 6,653,072, 6,686,515,
6,703,240, 6,716,631, 6,825,001, 6,902,922, 6,917,882, 6,946,296,
6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054,
7,220,566, 7,288,375, 7,384,387, 7,421,347, 7,430,477, 7,462,469,
7,534,564, 7,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391,
7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410,
7,868,138, 7,783,428, 7,873,477, 7,873,499, 7,904,249, 7,957,912,
7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138,
8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903,
8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, and all
related US and non-US counterparts: Ling et al., Anal. Biochem.,
254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74
[1996]1 Smith, Ann. Rev. Genet, 19:423-462 [1985]; Botstein et al.,
Science, 229:1193-1201 [1985] Carter, Biochem. J., 237:1-7 Kramer
et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323
[1985] Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999];
Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et
al., Nature, 391:288-291 [1998]1 Crameri, et al., Nat. Biotechnol.,
15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,
94:4504-4509 [1997] Crameri et al., Nat. Biotechnol., 14:315-319
[1996]; Stemmer, Nature, 370:389-391 [1994] Stemmer, Proc. Nat.
Acad. Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO
97/35966; WO 98/27230: WO 00/42651; WO 01/75767; and WO
2009/152336, all of which are incorporated herein by
reference).
In some embodiments, the variant transglutaminase of the present
invention further comprise additional sequences that do not alter
the encoded activity of the enzyme. For example, in some
embodiments, the variant transglutaminase are linked to an epitope
tag or to another sequence useful in purification.
In some embodiments, the variant transglutaminase polypeptides of
the present invention are secreted from the host cell in which they
are expressed (e.g., a yeast or filamentous fungal host cell) and
are expressed as a pre-protein including a signal peptide (i.e., an
amino acid sequence linked to the amino terminus of a polypeptide
and which directs the encoded polypeptide into the cell secretory
pathway).
In some embodiments, the signal peptide is an endogenous S.
mobaraensis transglutaminase signal peptide. In some additional
embodiments, signal peptides from other S. mobaraensis secreted
proteins are used. In some embodiments, other signal peptides find
use, depending on the host cell and other factors. Effective signal
peptide coding regions for filamentous fungal host cells include,
but are not limited to, the signal peptide coding regions obtained
from Aspergillus oryzae TAKA amylase, Aspergillus niger neutral
amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic
proteinase, Humicola insolens cellulase, Humicola lanuginosa
lipase, and T. reesei cellobiohydrolase II. Signal peptide coding
regions for bacterial host cells include, but are not limited to
the signal peptide coding regions obtained from the genes for
Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus
alpha-amylase, Bacillus licheniformis subtilisin, Bacillus
licheniformis .beta.-lactamase, Bacillus stearothermophilus neutral
proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. In some
additional embodiments, other signal peptides find use in the
present invention (See e.g., Simonen and Palva, Microbiol. Rev.,
57: 109-137 [1993], incorporated herein by reference). Additional
useful signal peptides for yeast host cells include those from the
genes for Saccharomyces cerevisiae alpha-factor, Saccharomyces
cerevisiae SUC2 invertase (See e.g. Taussig and Carlson, Nucl.
Acids Res., 11:1943-54 [1983]; SwissProt Accession No. P00724; and
Romanos et al., Yeast 8:423-488 [1992]). In some embodiments,
variants of these signal peptides and other signal peptides find
use. Indeed, it is not intended that the present invention be
limited to any specific signal peptide, as any suitable signal
peptide known in the art finds use in the present invention.
In some embodiments, the present invention provides polynucleotides
encoding variant transglutaminase polypeptides, and/or biologically
active fragments thereof, as described herein. In some embodiments,
the polynucleotide is operably linked to one or more heterologous
regulatory or control sequences that control gene expression to
create a recombinant polynucleotide capable of expressing the
polypeptide. In some embodiments, expression constructs containing
a heterologous polynucleotide encoding a variant transglutaminase
is introduced into appropriate host cells to express the variant
transglutaminase.
Those of ordinary skill in the art understand that due to the
degeneracy of the genetic code, a multitude of nucleotide sequences
encoding variant transglutaminase polypeptides of the present
invention exist. For example, the codons AGA, AGG, CGA, CGC, CGG,
and CGU all encode the amino acid arginine. Thus, at every position
in the nucleic acids of the invention where an arginine is
specified by a codon, the codon can be altered to any of the
corresponding codons described above without altering the encoded
polypeptide. It is understood that "U" in an RNA sequence
corresponds to "T" in a DNA sequence. The invention contemplates
and provides each and every possible variation of nucleic acid
sequence encoding a polypeptide of the invention that could be made
by selecting combinations based on possible codon choices.
As indicated above, DNA sequence encoding a transglutaminase may
also be designed for high codon usage bias codons (codons that are
used at higher frequency in the protein coding regions than other
codons that code for the same amino acid). The preferred codons may
be determined in relation to codon usage in a single gene, a set of
genes of common function or origin, highly expressed genes, the
codon frequency in the aggregate protein coding regions of the
whole organism, codon frequency in the aggregate protein coding
regions of related organisms, or combinations thereof. A codon
whose frequency increases with the level of gene expression is
typically an optimal codon for expression. In particular, a DNA
sequence can be optimized for expression in a particular host
organism. A variety of methods are well-known in the art for
determining the codon frequency (e.g., codon usage, relative
synonymous codon usage) and codon preference in specific organisms,
including multivariate analysis (e.g., using cluster analysis or
correspondence analysis,) and the effective number of codons used
in a gene. The data source for obtaining codon usage may rely on
any available nucleotide sequence capable of coding for a protein.
These data sets include nucleic acid sequences actually known to
encode expressed proteins (e.g., complete protein coding
sequences-CDS), expressed sequence tags (ESTs), or predicted coding
regions of genomic sequences, as is well-known in the art.
Polynucleotides encoding variant transglutaminases can be prepared
using any suitable methods known in the art. Typically,
oligonucleotides are individually synthesized, then joined (e.g.,
by enzymatic or chemical ligation methods, or polymerase-mediated
methods) to form essentially any desired continuous sequence. In
some embodiments, polynucleotides of the present invention are
prepared by chemical synthesis using, any suitable methods known in
the art, including but not limited to automated synthetic methods.
For example, in the phosphoramidite method, oligonucleotides are
synthesized (e.g., in an automatic DNA synthesizer), purified,
annealed, ligated and cloned in appropriate vectors. In some
embodiments, double stranded DNA fragments are then obtained either
by synthesizing the complementary strand and annealing the strands
together under appropriate conditions, or by adding the
complementary strand using DNA polymerase with an appropriate
primer sequence. There are numerous general and standard texts that
provide methods useful in the present invention are well known to
those skilled in the art.
The engineered transglutaminases can be obtained by subjecting the
polynucleotide encoding the naturally occurring transglutaminase to
mutagenesis and/or directed evolution methods, as discussed above.
Mutagenesis may be performed in accordance with any of the
techniques known in the art, including random and site-specific
mutagenesis. Directed evolution can be performed with any of the
techniques known in the art to screen for improved variants
including shuffling. Other directed evolution procedures that find
use include, but are not limited to staggered extension process
(StEP), in vitro recombination, mutagenic PCR, cassette
mutagenesis, splicing by overlap extension (SOEing), ProSAR.TM.
directed evolution methods, etc., as well as any other suitable
methods.
The clones obtained following mutagenesis treatment are screened
for engineered transglutaminases having a desired improved enzyme
property. Measuring enzyme activity from the expression libraries
can be performed using the standard biochemistry technique of
monitoring the rate of product formation. Where an improved enzyme
property desired is thermal stability, enzyme activity may be
measured after subjecting the enzyme preparations to a defined
temperature and measuring the amount of enzyme activity remaining
after heat treatments. Clones containing a polynucleotide encoding
a transglutaminase are then isolated, sequenced to identify the
nucleotide sequence changes (if any), and used to express the
enzyme in a host cell.
When the sequence of the engineered polypeptide is known, the
polynucleotides encoding the enzyme can be prepared by standard
solid-phase methods, according to known synthetic methods. In some
embodiments, fragments of up to about 100 bases can be individually
synthesized, then joined (e.g., by enzymatic or chemical ligation
methods, or polymerase mediated methods) to form any desired
continuous sequence. For example, polynucleotides and
oligonucleotides of the invention can be prepared by chemical
synthesis (e.g., using the classical phosphoramidite method
described by Beaucage et al., Tet. Lett., 22:1859-69 [1981], or the
method described by Matthes et al., EMBO J., 3:801-05 [1984], as it
is typically practiced in automated synthetic methods). According
to the phosphoramidite method, oligonucleotides are synthesized
(e.g., in an automatic DNA synthesizer), purified, annealed,
ligated and cloned in appropriate vectors. In addition, essentially
any nucleic acid can be obtained from any of a variety of
commercial sources (e.g., The Midland Certified Reagent Company,
Midland, Tex., The Great American Gene Company, Ramona, Calif.,
ExpressGen Inc. Chicago, Ill., Operon Technologies Inc., Alameda,
Calif., and many others).
The present invention also provides recombinant constructs
comprising a sequence encoding at least one variant
transglutaminase, as provided herein. In some embodiments, the
present invention provides an expression vector comprising a
variant transglutaminase polynucleotide operably linked to a
heterologous promoter. In some embodiments, expression vectors of
the present invention are used to transform appropriate host cells
to permit the host cells to express the variant transglutaminase
protein. Methods for recombinant expression of proteins in fungi
and other organisms are well known in the art, and a number of
expression vectors are available or can be constructed using
routine methods. In some embodiments, nucleic acid constructs of
the present invention comprise a vector, such as, a plasmid, a
cosmid, a phage, a virus, a bacterial artificial chromosome (BAC),
a yeast artificial chromosome (YAC), and the like, into which a
nucleic acid sequence of the invention has been inserted. In some
embodiments, polynucleotides of the present invention are
incorporated into any one of a variety of expression vectors
suitable for expressing variant transglutaminase polypeptide(s).
Suitable vectors include, but are not limited to chromosomal,
nonchromosomal and synthetic DNA sequences (e.g., derivatives of
SV40), as well as bacterial plasmids, phage DNA, baculovirus, yeast
plasmids, vectors derived from combinations of plasmids and phage
DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,
pseudorabies, adenovirus, adeno-associated virus, retroviruses, and
many others. Any suitable vector that transduces genetic material
into a cell, and, if replication is desired, which is replicable
and viable in the relevant host finds use in the present
invention.
In some embodiments, the construct further comprises regulatory
sequences, including but not limited to a promoter, operably linked
to the protein encoding sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art. Indeed, in
some embodiments, in order to obtain high levels of expression in a
particular host it is often useful to express the variant
transglutaminases of the present invention under the control of a
heterologous promoter. In some embodiments, a promoter sequence is
operably linked to the 5' region of the variant transglutaminase
coding sequence using any suitable method known in the art.
Examples of useful promoters for expression of variant
transglutaminases include, but are not limited to promoters from
fungi. In some embodiments, a promoter sequence that drives
expression of a gene other than a transglutaminase gene in a fungal
strain finds use. As a non-limiting example, a fungal promoter from
a gene encoding an endoglucanase may be used. In some embodiments,
a promoter sequence that drives the expression of a
transglutaminase gene in a fungal strain other than the fungal
strain from which the transglutaminases were derived finds use.
Examples of other suitable promoters useful for directing the
transcription of the nucleotide constructs of the present invention
in a filamentous fungal host cell include, but are not limited to
promoters obtained from the genes for Aspergillus oryzae TAKA
amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger
neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, and Fusarium oxysporum trypsin-like protease (See
e.g., WO 96/00787, incorporated herein by reference), as well as
the NA2-tpi promoter (a hybrid of the promoters from the genes for
Aspergillus niger neutral alpha-amylase and Aspergillus oryzae
triose phosphate isomerase), promoters such as cbh1, cbh2, egl1,
egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (See e.g., Nunberg et
al., Mol. Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBO J.,
3:1581-85 [1984]; and European Patent Appln. 137280, all of which
are incorporated herein by reference), and mutant, truncated, and
hybrid promoters thereof.
In yeast host cells, useful promoters include, but are not limited
to those from the genes for Saccharomyces cerevisiae enolase
(eno-1), Saccharomyces cerevisiae galactokinase (gall),
Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP),
and S. cerevisiae 3-phosphoglycerate kinase. Additional useful
promoters useful for yeast host cells are known in the art (See
e.g., Romanos et al., Yeast 8:423-488 [1992], incorporated herein
by reference). In addition, promoters associated with chitinase
production in fungi find use in the present invention (See e.g.,
Blaiseau and Lafay, Gene 120243-248 [1992]; and Limon et al., Curr.
Genet., 28:478-83 [1995], both of which are incorporated herein by
reference).
For bacterial host cells, suitable promoters for directing
transcription of the nucleic acid constructs of the present
disclosure, include but are not limited to the promoters obtained
from the E. coli lac operon, E. coli trp operon, bacteriophage
lambda, Streptomyces coelicolor agarase gene (dagA), Bacillus
subtilis levansucrase gene (sacB), Bacillus licheniformis
alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic
amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene
(amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus
subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene
(See e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75:
3727-3731 [1978]), as well as the tac promoter (See e.g., DeBoer et
al., Proc. Natl. Acad. Sci. USA 80: 21-25 [1983]).
In some embodiments, cloned variant transglutaminases of the
present invention also have a suitable transcription terminator
sequence, a sequence recognized by a host cell to terminate
transcription. The terminator sequence is operably linked to the 3'
terminus of the nucleic acid sequence encoding the polypeptide. Any
terminator that is functional in the host cell of choice finds use
in the present invention. Exemplary transcription terminators for
filamentous fungal host cells include, but are not limited to those
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease (See e.g., U.S. Pat. No. 7,399,627,
incorporated herein by reference). In some embodiments, exemplary
terminators for yeast host cells include those obtained from the
genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are well-known to those skilled in the art
(See e.g., Romanos et al., Yeast 8:423-88 L19921).
In some embodiments, a suitable leader sequence is part of a cloned
variant transglutaminase sequence, which is a nontranslated region
of an mRNA that is important for translation by the host cell. The
leader sequence is operably linked to the 5' terminus of the
nucleic acid sequence encoding the polypeptide. Any leader sequence
that is functional in the host cell of choice finds use in the
present invention. Exemplary leaders for filamentous fungal host
cells include, but are not limited to those obtained from the genes
for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose
phosphate isomerase. Suitable leaders for yeast host cells include,
but are not limited to those obtained from the genes for
Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae
3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor,
and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
In some embodiments, the sequences of the present invention also
comprise a polyadenylation sequence, which is a sequence operably
linked to the 3' terminus of the nucleic acid sequence and which,
when transcribed, is recognized by the host cell as a signal to add
polyadenosine residues to transcribed mRNA. Any polyadenylation
sequence which is functional in the host cell of choice finds use
in the present invention. Exemplary polyadenylation sequences for
filamentous fungal host cells include, but are not limited to those
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus
niger alpha-glucosidase. Useful polyadenylation sequences for yeast
host cells are known in the art (See e.g., Guo and Sherman, Mol.
Cell. Biol., 15:5983-5990 [1995]).
In some embodiments, the control sequence comprises a signal
peptide coding region encoding an amino acid sequence linked to the
amino terminus of a polypeptide and directs the encoded polypeptide
into the cell's secretory pathway. The 5' end of the coding
sequence of the nucleic acid sequence may inherently contain a
signal peptide coding region naturally linked in translation
reading frame with the segment of the coding region that encodes
the secreted polypeptide. Alternatively, the 5' end of the coding
sequence may contain a signal peptide coding region that is foreign
to the coding sequence. The foreign signal peptide coding region
may be required where the coding sequence does not naturally
contain a signal peptide coding region.
Alternatively, the foreign signal peptide coding region may simply
replace the natural signal peptide coding region in order to
enhance secretion of the polypeptide. However, any signal peptide
coding region which directs the expressed polypeptide into the
secretory pathway of a host cell of choice may be used in the
present invention.
Effective signal peptide coding regions for bacterial host cells
include, but are not limited to the signal peptide coding regions
obtained from the genes for Bacillus NClB 11837 maltogenic amylase,
Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis
subtilisin. Bacillus licheniformis beta-lactamase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are known in the
art (See e.g., Simonen and Palva. Microbiol. Rev., 57: 109-137
[1993]).
Effective signal peptide coding regions for filamentous fungal host
cells include, but are not limited to the signal peptide coding
regions obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger neutral amylase, Aspergillus niger
glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola
insolens cellulase, and Humicola lanuginosa lipase.
Useful signal peptides for yeast host cells include, but are not
limited to genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding regions are known in the art (See e.g., Romanos et al.,
[1992], supra).
In some embodiments, the control sequence comprises a propeptide
coding region that codes for an amino acid sequence positioned at
the amino terminus of a polypeptide. The resultant polypeptide is
known as a proenzyme or propolypeptide (or a zymogen in some
cases). A propolypeptide is generally inactive and can be converted
to a mature active transglutaminase polypeptide by catalytic or
autocatalytic cleavage of the propeptide from the propolypeptide.
The propeptide coding region may be obtained from the genes for
Bacillus subtilis alkaline protease (aprE), Bacillus subtilis
neutral protease (nprT), Saccharomyces cerevisiae alpha-factor,
Rhizomucor miehei aspartic proteinase, and Myceliophthora
thermophila lactase (See e.g., WO 95/33836).
Where both signal peptide and propeptide regions are present at the
amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
In some embodiments, regulatory sequences are also used to allow
the regulation of the expression of the polypeptide relative to the
growth of the host cell. Examples of regulatory systems are those
which cause the expression of the gene to be turned on or off in
response to a chemical or physical stimulus, including the presence
of a regulatory compound. In prokaryotic host cells, suitable
regulatory sequences include, but are not limited to the lac, tac,
and trp operator systems. In yeast host cells, suitable regulatory
systems include, as examples, the ADH2 system or GAL1 system. In
filamentous fungi, suitable regulatory sequences include the TAKA
alpha-amylase promoter, Aspergillus niger glucoamylase promoter,
and Aspergillus oryzae glucoamylase promoter.
Other examples of regulatory sequences are those which allow for
gene amplification. In eukaryotic systems, these include the
dihydrofolate reductase gene, which is amplified in the presence of
methotrexate, and the metallothionein genes, which are amplified
with heavy metals. In these cases, the nucleic acid sequence
encoding the transglutaminase polypeptide of the present invention
would be operably linked with the regulatory sequence.
Thus, in additional embodiments, the present invention provides
recombinant expression vectors comprising a polynucleotide encoding
an engineered transglutaminase polypeptide or a variant thereof,
and one or more expression regulating regions such as a promoter
and a terminator, a replication origin, etc., depending on the type
of hosts into which they are to be introduced. In some embodiments,
the various nucleic acid and control sequences described above are
joined together to produce a recombinant expression vector that may
include one or more convenient restriction sites to allow for
insertion or substitution of the nucleic acid sequence encoding the
polypeptide at such sites. Alternatively, in some embodiments, the
nucleic acid sequences are expressed by inserting the nucleic acid
sequence or a nucleic acid construct comprising the sequence into
an appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the
coding sequence is operably linked with the appropriate control
sequences for expression.
The recombinant expression vector comprises any suitable vector
(e.g., a plasmid or virus), that can be conveniently subjected to
recombinant DNA procedures and can bring about the expression of
the polynucleotide sequence. The choice of the vector typically
depends on the compatibility of the vector with the host cell into
which the vector is to be introduced. In some embodiments, the
vectors are linear or closed circular plasmids.
In some embodiments, the expression vector is an autonomously
replicating vector (i.e., a vector that exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, such as a plasmid, an extrachromosomal
element, a minichromosome, or an artificial chromosome). In some
embodiments, the vector contains any means for assuring
self-replication. Alternatively, in some other embodiments, upon
being introduced into the host cell, the vector is integrated into
the genome and replicated together with the chromosome(s) into
which it has been integrated. Furthermore, in additional
embodiments, a single vector or plasmid or two or more vectors or
plasmids which together contain the total DNA to be introduced into
the genome of the host cell, or a transposon find use.
In some embodiments, the expression vector of the present invention
contains one or more selectable markers, which permit easy
selection of transformed cells. A "selectable marker" is a gene,
the product of which provides for biocide or viral resistance,
resistance to antimicrobials or heavy metals, prototrophy to
auxotrophs, and the like. Any suitable selectable markers for use
in a filamentous fungal host cell find use in the present
invention, including, but are not limited to, amdS (acetamidase),
argB (omithine carbamoyltransferase), bar (phosphinothricin
acetyltransferase), hph (hygromycin phosphotransferase), niaD
(nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase),
sC (sulfate adenyltransferase), and trpC (anthranilate synthase),
as well as equivalents thereof. Additional markers useful in host
cells such as Aspergillus, include but are not limited to the amdS
and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and
the bar gene of Streptomyces hygroscopicus. Suitable markers for
yeast host cells include, but are not limited to ADE2, HIS3, LEU2,
LYS2, MET3, TRP1, and URA3. Examples of bacterial selectable
markers include, but are not limited to the dal genes from Bacillus
subtilis or Bacillus licheniformis, or markers, which confer
antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol, and or tetracycline resistance.
In some embodiments, the expression vectors of the present
invention contain an element(s) that permits integration of the
vector into the host cell's genome or autonomous replication of the
vector in the cell independent of the genome. In some embodiments
involving integration into the host cell genome, the vectors rely
on the nucleic acid sequence encoding the polypeptide or any other
element of the vector for integration of the vector into the genome
by homologous or nonhomologous recombination.
In some alternative embodiments, the expression vectors contain
additional nucleic acid sequences for directing integration by
homologous recombination into the genome of the host cell. The
additional nucleic acid sequences enable the vector to be
integrated into the host cell genome at a precise location(s) in
the chromosome(s). To increase the likelihood of integration at a
precise location, the integrational elements preferably contain a
sufficient number of nucleotides, such as 100 to 10,000 base pairs,
preferably 400 to 10,000 base pairs, and most preferably 800 to
10,000 base pairs, which are highly homologous with the
corresponding target sequence to enhance the probability of
homologous recombination. The integrational elements may be any
sequence that is homologous with the target sequence in the genome
of the host cell. Furthermore, the integrational elements may be
non-encoding or encoding nucleic acid sequences. On the other hand,
the vector may be integrated into the genome of the host cell by
non-homologous recombination.
For autonomous replication, the vector may further comprise an
origin of replication enabling the vector to replicate autonomously
in the host cell in question. Examples of bacterial origins of
replication are P15A ori or the origins of replication of plasmids
pBR322, pUC 19, pACYC177 (which plasmid has the P15A ori), or
pACYC184 permitting replication in E. coli, and pUB 110, pE 194,
pTA1060, or pAM.quadrature.1 permitting replication in Bacillus.
Examples of origins of replication for use in a yeast host cell are
the 2 micron origin of replication, ARS1, ARS4, the combination of
ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of
replication may be one having a mutation which makes it's
functioning temperature-sensitive in the host cell (See e.g.,
Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433 [1978]).
In some embodiments, more than one copy of a nucleic acid sequence
of the present invention is inserted into the host cell to increase
production of the gene product. An increase in the copy number of
the nucleic acid sequence can be obtained by integrating at least
one additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the nucleic
acid sequence where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
nucleic acid sequence, can be selected for by cultivating the cells
in the presence of the appropriate selectable agent.
Many of the expression vectors for use in the present invention are
commercially available. Suitable commercial expression vectors
include, but are not limited to the p3xFLAG.TM. expression vectors
(Sigma-Aldrich Chemicals), which include a CMV promoter and hGH
polyadenylation site for expression in mammalian host cells and a
pBR322 origin of replication and ampicillin resistance markers for
amplification in E. coli. Other suitable expression vectors
include, but are not limited to pBluescriptII SK(-) and pBK-CMV
(Stratagene), and plasmids derived from pBR322 (Gibco BRL), pUC
(Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (See e.g., Lathe et
al., Gene 57:193-201 [1987]).
Thus, in some embodiments, a vector comprising a sequence encoding
at least one variant transglutaminase is transformed into a host
cell in order to allow propagation of the vector and expression of
the variant transglutaminase(s). In some embodiments, the variant
transglutaminases are post-translationally modified to remove the
signal peptide and in some cases may be cleaved after secretion. In
some embodiments, the transformed host cell described above is
cultured in a suitable nutrient medium under conditions permitting
the expression of the variant transglutaminase(s). Any suitable
medium useful for culturing the host cells finds use in the present
invention, including, but not limited to minimal or complex media
containing appropriate supplements. In some embodiments, host cells
are grown in HTP media. Suitable media are available from various
commercial suppliers or may be prepared according to published
recipes (e.g., in catalogues of the American Type Culture
Collection).
In another aspect, the present invention provides host cells
comprising a polynucleotide encoding an improved transglutaminase
polypeptide provided herein, the polynucleotide being operatively
linked to one or more control sequences for expression of the
transglutaminase enzyme in the host cell. Host cells for use in
expressing the transglutaminase polypeptides encoded by the
expression vectors of the present invention are well known in the
art and include but are not limited to, bacterial cells, such as E.
coli, Bacillus megaterium, Lactobacillus kefir, Streptomyces and
Salmonella typhimurium cells; fungal cells, such as yeast cells
(e.g, Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession
No. 201178)): insect cells such as Drosophila S2 and Spodoptera Sf9
cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma
cells; and plant cells. Appropriate culture media and growth
conditions for the above-described host cells are well known in the
art.
Polynucleotides for expression of the transglutaminase may be
introduced into cells by various methods known in the art.
Techniques include among others, electroporation, biolistic
particle bombardment, liposome mediated transfection, calcium
chloride transfection, and protoplast fusion. Various methods for
introducing polynucleotides into cells are known to those skilled
in the art.
In some embodiments, the host cell is a eukaryotic cell. Suitable
eukaryotic host cells include, but are not limited to, fungal
cells, algal cells, insect cells, and plant cells. Suitable fungal
host cells include, but are not limited to, Ascomycota,
Basidiomycota, Deuteromycota, Zygomycota, Fungi imperfecti. In some
embodiments, the fungal host cells are yeast cells and filamentous
fungal cells.
The filamentous fungal host cells of the present invention include
all filamentous forms of the subdivision Eumycotina and Oomycota.
Filamentous fungi are characterized by a vegetative mycelium with a
cell wall composed of chitin, cellulose and other complex
polysaccharides. The filamentous fungal host cells of the present
invention are morphologically distinct from yeast.
In some embodiments of the present invention, the filamentous
fungal host cells are of any suitable genus and species, including,
but not limited to Achlya, Acremonmum, Aspergillus, Aureobasidium,
Bjerkandera, Ceriporiopsis, Cephalosporium, Chrysosporium,
Cochliobolus, Corynascus, Crvphonectria, Crvptococcus, Coprinus,
Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium,
Humicola, Hypocrea, Myceliophihora, Mucor, Neurospora, Penicillium,
Podospora, Phlebia, Piromyces, Pyriculana, Rhizomucor, Rhizopus,
Schizophyllum, Scytahdium, Sporotrichum, Talaromyces, Thermoascus,
Thielavia, Trametes, Tolvpocladium, Trichoderma, Verticillium,
and/or Volvariella, and/or teleomorphs, or anamorphs, and synonyms,
basionyms, or taxonomic equivalents thereof.
In some embodiments of the present invention, the host cell is a
yeast cell, including but not limited to cells of Candida,
Hansenula, Saccharomyces, Schizosaccharomyces, Pichia,
Kluyveromyces, or Yarrowia species. In some embodiments of the
present invention, the yeast cell is Hansenula polymorpha.
Saccharomyces cerevisiae, Saccharomyces carlsbergensis,
Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces
kluyver, Schizosaccharomvces pombe, Pichia pastoris, Pichia
finlandica, Pichia trehalophila, Pichia kodamae, Pichia
membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia
salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis,
Pichia methanolica, Pichia angusta, Kluyveromyces klctis, Candida
albicans, or Yarrowia lipolytica.
In some embodiments of the invention, the host cell is an algal
cell such as Chlamydomonas (e.g., C. reinhardtii) and Phormidium
(P. sp. ATCC29409).
In some other embodiments, the host cell is a prokaryotic cell.
Suitable prokaryotic cells include, but are not limited to
Gram-positive, Gram-negative and Gram-variable bacterial cells. Any
suitable bacterial organism finds use in the present invention,
including but not limited to Agrobacterium, Alicyclobacillus,
Anabaena, Anacystis. Acinetobacter, Acidothermus, Arthrobacter,
Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio,
Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium,
Chromatium, Coprcoccus, Escherichia, Enterococcus, Enterobacter,
Erwinia, Fusobactertum, Faecalibacterium, Francisella,
Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Klebsiella,
Lactobacillus, Lactococcus, Ilyobacter, Micrococcus,
Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium,
Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus,
Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburta,
Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces,
Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus,
Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma,
Tularensis, Temecula, Thermosynechococcus, Thermococcus,
Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. In some
embodiments, the host cell is a species of Agrobacterium,
Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera,
Geobacillus, Campylobacter, Clostridium, Corynebacterium,
Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus,
Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella,
Streptococcus, Streptomyces, or Zymomonas. In some embodiments, the
bacterial host strain is non-pathogenic to humans. In some
embodiments the bacterial host strain is an industrial strain.
Numerous bacterial industrial strains are known and suitable in the
present invention. In some embodiments of the present invention,
the bacterial host cell is an Agrobacterium species (e.g., A.
radiobacter, A. rhizogenes, and A. rubi). In some embodiments of
the present invention, the bacterial host cell is an Arthrobacter
species (e.g., A. aurescens, A. citreus, A. globiformis, A.
hydrocarboglutamicus, A. mysorens, A. nicotianae, A. parafineus, A.
protophonniae, A. roseoparqffinus, A. sulfireus, and A.
ureafaciens). In some embodiments of the present invention, the
bacterial host cell is a Bacillus species (e.g., B. thuringensis,
B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans,
B. pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B.
alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B.
halodurans, and B. amyloliquefaciens). In some embodiments, the
host cell is an industrial Bacillus strain including but not
limited to B. subtilis, B. pumilus, B. licheniformis, B.
megaterium. B. clausii, B. stearothermophilus, or B.
amyloliquefaciens. In some embodiments, the Bacillus host cells are
B. subtilis, B. licheniformis, B. megaterium, B.
stearothermophilus, and/or B. amyloliquefaciens. In some
embodiments, the bacterial host cell is a Clostridium species
(e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C.
saccharobutylicum, C. perfringens, and C. beijerinckii). In some
embodiments, the bacterial host cell is a Corynebacterium species
(e.g., C. glutamicum and C. acetoacidophilum). In some embodiments
the bacterial host cell is an Escherichia species (e.g., E. coli).
In some embodiments, the host cell is Escherichia coli W3110. In
some embodiments, the bacterial host cell is an Erwinia species
(e.g., E. uredovora, E. carotovora, E. ananas, E. herbicola, E.
punctata, and E. terreus). In some embodiments, the bacterial host
cell is a Pantoea species (e.g., P. citrea, and P. agglomerans). In
some embodiments the bacterial host cell is a Pseudomonas species
(e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10).
In some embodiments, the bacterial host cell is a Streptococcus
species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some
embodiments, the bacterial host cell is a Streptomyces species
(e.g., S. ambofaciens, S. achromogenes, S. avermrtilis, S.
coelicolor, S. aureofaciens. S. aureus, S. fimgicidicus, S.
griseus, and S. lividans). In some embodiments, the bacterial host
cell is a Zymomonas species (e.g., Z. mobilis, and Z
lipolytica).
Many prokaryotic and eukaryotic strains that find use in the
present invention are readily available to the public from a number
of culture collections such as American Type Culture Collection
(ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
(DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural
Research Service Patent Culture Collection, Northern Regional
Research Center (NRRL).
In some embodiments, host cells are genetically modified to have
characteristics that improve protein secretion, protein stability
and/or other properties desirable for expression and/or secretion
of a protein. Genetic modification can be achieved by genetic
engineering techniques and/or classical microbiological techniques
(e.g., chemical or UV mutagenesis and subsequent selection).
Indeed, in some embodiments, combinations of recombinant
modification and classical selection techniques are used to produce
the host cells. Using recombinant technology, nucleic acid
molecules can be introduced, deleted, inhibited or modified, in a
manner that results in increased yields of transglutaminase
variant(s) within the host cell and/or in the culture medium. For
example, knockout of Alp1 function results in a cell that is
protease deficient, and knockout of pyr5 function results in a cell
with a pyrimidine deficient phenotype. In one genetic engineering
approach, homologous recombination is used to induce targeted gene
modifications by specifically targeting a gene in vivo to suppress
expression of the encoded protein. In alternative approaches,
siRNA, antisense and/or ribozyme technology find use in inhibiting
gene expression. A variety of methods are known in the art for
reducing expression of protein in cells, including, but not limited
to deletion of all or part of the gene encoding the protein and
site-specific mutagenesis to disrupt expression or activity of the
gene product. (See e.g., Chaveroche et al., Nucl. Acids Res., 28:22
e97 [2000]; Cho et al., Molec. Plant Microbe Interact., 19:7-15
[2006]; Maruyama and Kitamoto, Biotechnol Lett. 30:1811-1817
[2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352 [2004];
and You et al., Arch. Micriobiol., 191:615-622 [2009], all of which
are incorporated by reference herein). Random mutagenesis, followed
by screening for desired mutations also finds use (See e.g.,
Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon
et al., Eukary. Cell 2:247-55 [2003], both of which are
incorporated by reference).
Introduction of a vector or DNA construct into a host cell can be
accomplished using any suitable method known in the art, including
but not limited to calcium phosphate transfection, DEAE-dextran
mediated transfection, PEG-mediated transformation,
electroporation, or other common techniques known in the art. In
some embodiments, the Escherichia coli expression vector pCK
100900i (See, US Pat. Appln. Publn. 2006/0195947, which is hereby
incorporated by reference herein) finds use.
In some embodiments, the engineered host cells (i.e., "recombinant
host cells") of the present invention are cultured in conventional
nutrient media modified as appropriate for activating promoters,
selecting transformants, or amplifying the transglutaminase
polynucleotide. Culture conditions, such as temperature, pH and the
like, are those previously used with the host cell selected for
expression, and are well-known to those skilled in the art. As
noted, many standard references and texts are available for the
culture and production of many cells, including cells of bacterial,
plant, animal (especially mammalian) and archebacterial origin.
In some embodiments, cells expressing the variant transglutaminase
polypeptides of the invention are grown under batch or continuous
fermentations conditions. Classical "batch fermentation" is a
closed system, wherein the compositions of the medium is set at the
beginning of the fermentation and is not subject to artificial
alternations during the fermentation. A variation of the batch
system is a "fed-batch fermentation" which also finds use in the
present invention. In this variation, the substrate is added in
increments as the fermentation progresses. Fed-batch systems are
useful when catabolite repression is likely to inhibit the
metabolism of the cells and where it is desirable to have limited
amounts of substrate in the medium. Batch and fed-batch
fermentations are common and well known in the art. "Continuous
fermentation" is an open system where a defined fermentation medium
is added continuously to a bioreactor and an equal amount of
conditioned medium is removed simultaneously for processing.
Continuous fermentation generally maintains the cultures at a
constant high density where cells are primarily in log phase
growth. Continuous fermentation systems strive to maintain steady
state growth conditions. Methods for modulating nutrients and
growth factors for continuous fermentation processes as well as
techniques for maximizing the rate of product formation are well
known in the art of industrial microbiology.
In some embodiments of the present invention, cell-free
transcription/translation systems find use in producing variant
transglutaminase(s). Several systems are commercially available and
the methods are well-known to those skilled in the art.
The present invention provides methods of making variant
transglutaminase polypeptides or biologically active fragments
thereof. In some embodiments, the method comprises: providing a
host cell transformed with a polynucleotide encoding an amino acid
sequence that comprises at least about 70% (or at least about 75%,
at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 96%, at least about 97%, at least
about 98%, or at least about 99%) sequence identity to SEQ ID NO:
2, 6, 34, and/or 256, and comprising at least one mutation as
provided herein; culturing the transformed host cell in a culture
medium under conditions in which the host cell expresses the
encoded variant transglutaminase polypeptide; and optionally
recovering or isolating the expressed variant transglutaminase
polypeptide, and/or recovering or isolating the culture medium
containing the expressed variant transglutaminase polypeptide. In
some embodiments, the methods further provide optionally lysing the
transformed host cells after expressing the encoded
transglutaminase polypeptide and optionally recovering and/or
isolating the expressed variant transglutaminase polypeptide from
the cell lysate. The present invention further provides methods of
making a variant transglutaminase polypeptide comprising
cultivating a host cell transformed with a variant transglutaminase
polypeptide under conditions suitable for the production of the
variant transglutaminase polypeptide and recovering the variant
transglutaminase polypeptide. Typically, recovery or isolation of
the transglutaminase polypeptide is from the host cell culture
medium, the host cell or both, using protein recovery techniques
that are well known in the art, including those described herein.
In some embodiments, host cells are harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification. Microbial cells employed
in expression of proteins can be disrupted by any convenient
method, including, but not limited to freeze-thaw cycling,
sonication, mechanical disruption, and/or use of cell lysing
agents, as well as many other suitable methods well known to those
skilled in the art.
Engineered transglutaminase enzymes expressed in a host cell can be
recovered from the cells and/or the culture medium using any one or
more of the techniques known in the art for protein purification,
including, among others, lysozyme treatment, sonication,
filtration, salting-out, ultra-centrifugation, and chromatography.
Suitable solutions for lysing and the high efficiency extraction of
proteins from bacteria, such as E. coli, are commercially available
under the trade name CelLytic B.TM. (Sigma-Aldrich). Thus, in some
embodiments, the resulting polypeptide is recovered/isolated and
optionally purified by any of a number of methods known in the art.
For example, in some embodiments, the polypeptide is isolated from
the nutrient medium by conventional procedures including, but not
limited to, centrifugation, filtration, extraction, spray-drying,
evaporation, chromatography (e.g., ion exchange, affinity,
hydrophobic interaction, chromatofocusing, and size exclusion), or
precipitation. In some embodiments, protein refolding steps are
used, as desired, in completing the configuration of the mature
protein. In addition, in some embodiments, high performance liquid
chromatography (HPLC) is employed in the final purification steps.
For example, in some embodiments, methods known in the art, find
use in the present invention (See e.g., Parry et al., Biochem. J.,
353:117 [2001], and Hong et al., Appl. Microbiol. Biotechnol.,
73:1331 [2007], both of which are incorporated herein by
reference). Indeed, any suitable purification methods known in the
art find use in the present invention.
Chromatographic techniques for isolation of the transglutaminase
polypeptide include, but are not limited to reverse phase
chromatography high performance liquid chromatography, ion exchange
chromatography, gel electrophoresis, and affinity chromatography.
Conditions for purifying a particular enzyme will depend, in part,
on factors such as net charge, hydrophobicity, hydrophilicity,
molecular weight, molecular shape, etc., are known to those skilled
in the art.
In some embodiments, affinity techniques find use in isolating the
improved transglutaminase enzymes. For affinity chromatography
purification, any antibody which specifically binds the
transglutaminase polypeptide may be used. For the production of
antibodies, various host animals, including but not limited to
rabbits, mice, rats, etc., may be immunized by injection with the
transglutaminase. The transglutaminase polypeptide may be attached
to a suitable carrier, such as BSA, by means of a side chain
functional group or linkers attached to a side chain functional
group.
Various adjuvants may be used to increase the immunological
response, depending on the host species, including but not limited
to Freund's (complete and incomplete), mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanin, dinitrophenol, and potentially useful human
adjuvants such as BCG (Bacillus Calmette Guerin) and
Corynebacterium parvum.
In some embodiments, the transglutaminase variants are prepared and
used in the form of cells expressing the enzymes, as crude
extracts, or as isolated or purified preparations. In some
embodiments, the transglutaminase variants are prepared as
lyophilisates, in powder form (e.g., acetone powders), or prepared
as enzyme solutions. In some embodiments, the transglutaminase
variants are in the form of substantially pure preparations.
In some embodiments, the transglutaminase polypeptides are attached
to any suitable solid substrate. Solid substrates include but are
not limited to a solid phase, surface, and/or membrane. Solid
supports include, but are not limited to organic polymers such as
polystyrene, polyethylene, polypropylene, polyfluoroethylene,
polyethylencoxy, and polyacrylamide, as well as co-polymers and
grafts thereof. A solid support can also be inorganic, such as
glass, silica, controlled pore glass (CPG), reverse phase silica or
metal, such as gold or platinum. The configuration of the substrate
can be in the form of beads, spheres, particles, granules, a gel, a
membrane or a surface. Surfaces can be planar, substantially
planar, or non-planar. Solid supports can be porous or non-porous,
and can have swelling or non-swelling characteristics. A solid
support can be configured in the form of a well, depression, or
other container, vessel, feature, or location. A plurality of
supports can be configured on an array at various locations,
addressable for robotic delivery of reagents, or by detection
methods and/or instruments.
In some embodiments, immunological methods are used to purify
transglutaminase variants. In one approach, antibody raised against
a variant transglutaminase polypeptide (e.g., against a polypeptide
comprising any of SEQ ID NO: 2, 6, 34, and/or 256 and/or an
immunogenic fragment thereof) using conventional methods is
immobilized on beads, mixed with cell culture media under
conditions in which the variant transglutaminase is bound, and
precipitated. In a related approach, immunochromatography finds
use.
In some embodiments, the variant transglutaminases are expressed as
a fusion protein including a non-enzyme portion. In some
embodiments, the variant transglutaminase sequence is fused to a
purification facilitating domain. As used herein, the term
"purification facilitating domain" refers to a domain that mediates
purification of the polypeptide to which it is fused. Suitable
purification domains include, but are not limited to metal
chelating peptides, histidine-tryptophan modules that allow
purification on immobilized metals, a sequence which binds
glutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to
an epitope derived from the influenza hemagglutinin protein; See
e.g., Wilson et al., Cell 37:767 [1984]), maltose binding protein
sequences, the FLAG epitope utilized in the FLAGS
extension/affinity purification system (e.g., the system available
from Immunex Corp), and the like. One expression vector
contemplated for use in the compositions and methods described
herein provides for expression of a fusion protein comprising a
polypeptide of the invention fused to a polyhistidine region
separated by an enterokinase cleavage site. The histidine residues
facilitate purification on IMIAC (immobilized metal ion affinity
chromatography; See e.g., Porath et al., Prot. Exp. Purif.,
3:263-281 [1992]) while the enterokinase cleavage site provides a
means for separating the variant transglutaminase polypeptide from
the fusion protein. pGEX vectors (Promega) may also be used to
express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
ligand-agarose beads (e.g., glutathione-agarose in the case of
GST-fusions) followed by elution in the presence of free
ligand.
EXPERIMENTAL
Various features and embodiments of the disclosure are illustrated
in the following representative examples, which are intended to be
illustrative, and not limiting.
In the experimental disclosure below, the following abbreviations
apply: ppm (parts per million); M (molar); mM (millimolar), uM and
.mu.M (micromolar); nM (nanomolar); mol (moles); gm and g (gram);
mg (milligrams); ug and .mu.g (micrograms); L and l (liter), ml and
mL (milliliter): cm (centimeters); mm (millimeters), um and .mu.m
(micrometers): sec. (seconds): min(s) (minute(s)); h(s) and hr(s)
(hour(s)); U (units); MW (molecular weight); rpm (rotations per
minute); .degree. C. (degrees Centigrade); RT (room temperature);
CDS (coding sequence); DNA (deoxyribonucleic acid); RNA
(ribonucleic acid); aa (amino acid): TB (Terrific Broth: 12 g/L
bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mM
potassium phosphate, pH 7.0, 1 mM MgSO.sub.4); LB (Luria Bertani
broth); CAM (chloramphenicol); PMBS (polymyxin B sulfate); IPTG
(isopropyl thiogalactoside); PEG (polyethylene glycol); TFA
(trifluoroacetic acid); CHES (2-cyclohexylamino)ethanesulfonic
acid; acetonitrile (MeCN); dimethylsulfoxide (DMSO);
dimethylacetamide (DMAc); HPLC (high performance liquid
chromatography); UPLC (ultra performance liquid chromatography);
FIOPC (fold improvement over positive control); HTP (high
throughput); MWD (multiple wavelength detector); UV (ultraviolet);
Codexis (Codexis. Inc., Redwood City, Calif.); Sigma-Aldrich
(Sigma-Aldrich, St. Louis, Mo.); Millipore (Millipore, Corp.,
Billerica Mass.); Difco (Difco Laboratories, BD Diagnostic Systems,
Detroit, Mich.); GeneOracle (GeneOracle, Santa Clara, Calif.); Boca
Scientific (Boca Scientific, Ind., Boca Raton, Fla.); Pall (Pall
Corporation, Port Washington, N.Y.); Vivaproducts (Vivaproducts,
Inc., Littleton, Mass.); Thermotron (Thermotron, Inc., Holland,
Mich.) Infors (Infors USA, Inc., Annapolis Junction, Md.); Genetix
(Genetic USA Inc., Beaverton, Oreg.); Daicel (Daicel, West Chester,
Pa.); Genetix (Genetix USA, Inc., Beaverton, Oreg.); Molecular
Devices (Molecular Devices, LLC, Sunnyvale, Calif.); Applied
Biosystems (Applied Biosystems, part of Life Technologies, Corp.,
Grand Island, N.Y.); Life Technologies (Life Technologies, Corp.,
Grand Island, N.Y.); Agilent (Agilent Technologies, Inc., Santa
Clara, Calif.); Thermo Scientific (part of Thermo Fisher
Scientific, Waltham, Mass.); (Infors: Infors-HT, Bottmingen/Basel,
Switzerland); Corning (Corning, Inc., Palo Alto, Calif.); and
Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.); Microfluidics
(Microfluidics Corp., Newton, Mass.); Waters (Waters Corp.,
Milford, Mass.).
Example 1
Wild Type Streptomyces mobaraensis Transglutaminase (MTG) Gene
Acquisition and Construction of Expression Vector
A pro-gene coding for Streptomyces mobaraensis transglutaminase
(MTCG) was codon optimized for expression in B. megaterium based on
the reported amino acid sequence (Shimonishi et al., J. Biol.
Chem., 268:11565-115720 [1993]). The gene was synthesized by
GenOracle and codon-optimized using their proprietary software. The
DNA was sequence verified. The pro-MTG gene was cloned behind a B.
megaterium "optimized" signal peptide plus a spacer region (6 bases
encoding amino acid residues threonine and serine into an E.
coli/B. megaterium shuttle vector pSSBm, using the BsrGI/NgoMIV
cloning sites. The vector pSSBm is a modified vector based on the
shuttle vector pMMI525 (Boca Scientific). The signal peptide and
pro-gene were under the control of an xlyose promoter (Pxyl)
regulated by the xylose repressor gene (xylR) present on the
shuttle vector. The vector contained the `rep U` origin of
replication for Bacillus and a tetracycline ampicillin resistance
marker. The vector also contained the pBR322 origin of replication
and an ampicillin resistance marker for maintenance in E. coli. The
resulting plasmid (pSSBm-pre-pro-MTG) was transformed by a standard
PEG-mediated method of DNA transfer into B. megaterium protoplasts.
The pre-pro-MTG sequence from the transformants was verified. The
polynucleotide sequence of the pre-pro-MTG that includes a B.
megaterium signal peptide was cloned into the shuttle pSSBm vector
and the sequence is provided in SEQ ID NO: 6, the sequence of the
pro-MTG with a C-terminus histidine purification tag comprises SEQ
ID NO: 2 and the sequence of the mature MTG comprises SEQ ID NO:
4.
Example 2
B. megaterium Shake Flask Procedure
A single microbial colony of B. megaterium containing a plasmid
with the pre-pro-MTG gene was inoculated into 3 ml Luria-Bertani
(LB) broth (0.01 g/L peptone from casein, 0.005 g/L yeast extract,
0.01 g/L sodium chloride) containing 10 .mu.g/mL tetracycline.
Cells were grown overnight for at least 16 hrs, at 37.degree. C.,
with shaking at 250 rpm. The culture was then diluted into 100 mL
A5 media (2 g/L (NH4).sub.2SO.sub.4, 3.5 g/L KH.sub.2HPO.sub.4, 7.3
g/L Na.sub.2HPO.sub.4, 1 g/L yeast extract, pH to 6.8), 100 .mu.L
of trace elements solution (49 g/L MnCl.sub.2.4H.sub.2O, 45 g/L
CaCl.sub.2, 2.5 g/L (NH.sub.4)Mo.sub.7.O.sub.24.H.sub.2O, 2.5 g/L
CoCl.sub.2.6H.sub.2O), 1.5 mL of 20% glucose, 150 g/L of IM
MgSO.sub.4, 100 .mu.L of 10 mg/mL tetracycline, 100 .mu.L of 2.5
g/L FeSO.sub.4.7H.sub.2O in a 1000 ml flask to an optical density
at 600 nm (OD.sub.600) of 0.2 and allowed to grow at 37.degree. C.
Expression of the pre-pro-MTG gene was induced with 0.5% xylose
(final concentration) when the OD.sub.600 of the culture was 0.6 to
0.8 and incubated overnight, for at least 16 hrs. Cells were
pelleted by centrifugation (4000 rpm, 15 min, 4.degree. C.). The
clear media supernatant containing the secreted mature MTG enzyme
was collected and 60 mL of the supernatant transferred into the top
cell of a Jumbosep concentrator (PES membrane, 3,000 MWCO pore
size; Pall). The supernatant was centrifuged at room temperature,
4000 rpm, until the volume became less than 20 mL (.about.45 min).
The filtrate was discarded and the remaining 20 mL of supernatant
were added to the concentrate to make up a final volume of 40 mL.
The centrifugation of the 40 mL was continued at room temperature,
4000 rpm until the volume reached .about.20 mL (.about.45 min). The
20 mL of 5.times. concentrate were transferred into a Vivaspin 20
concentrator (PES membrane, 10,000 MWCO pore size, Vivaproducts),
and centrifuged until the volume was .about.1 mL (.about.60 min).
Then, 50 mM NaOAc buffer, pH=5.0 was added up to 20 mL volume
(first buffer exchange), and centrifugation continued at room
temperature, 4000 rpm until the volume was .about.1 mL (.about.60
min). Then, 50 mM NaOAc buffer pH=5.0 was added up to 20 mL volume
(second buffer exchange), and centrifugation continued at room
temperature, 4000 rpm until the volume was .about.5 mL (.about.60
min). The 20.times. concentrate was mixed well and stored and
stored at -20.degree. C. MTG activity was confirmed using the
hydroxymate assay and the insulin assays described herein.
Example 3
B. megaterium High Throughput Assays to Identify Improved MTG
Variants
Plasmid libraries containing variant pre-pro-MTG genes were
transformed into B. megaterium and plated on Luria-Bertani (LB)
agar plates containing 3 .mu.g/mL tetracycline with a DM3
regeneration media (400 mM sodium succinate dibasic, pH 7.3, 0.5%
casamino acids, 0.5% yeast extract, 0.4% K.sub.2HPO.sub.4, 0.2%
KH.sub.2PO.sub.4, 20 mM MgCl.sub.2, 0.5% glucose and 0.2% BSA)
overlay. After incubation for at least 18 hours at 30.degree. C.,
colonies were picked using a Q-bot.RTM. robotic colony picker
(Genetix) into shallow, 96-well well microtiter plates containing
180 .mu.L LB and 10 .mu.g/mL tetracycline. Cells were grown
overnight at 37.degree. C. with shaking at 200 rpm and 85%
humidity. Then, 20 .mu.L of this culture were transferred into
96-well microtiter plates (deep well) containing 380 .mu.L of
subculture media (A5 0.3% glucose medium, as described in Example
2), with 10 .mu.g/mL tetracycline, 1% xylose and 0.25 mM
ZnSO.sub.4. The plates were then incubated at 37.degree. C. with
shaking at 250 rpm and 85% humidity for approximately 15-18 hours.
These plates were then centrifuged at 4000 rpm for 15 minutes and
the clear media supernatant containing the secreted mature MTG
enzyme was used for the high throughput hydroxymate assay.
Example 4
E. coli Expression Hosts Containing Recombinant TG Genes
The initial transglutaminase (TG) parent enzyme (SEQ ID NO: 6) of
the present invention was codon optimized for expression in E.
coli, synthesized and cloned into a pCK 110900 vector (See e.g.,
See, U.S. Pat. No. 7,629,157 and US Pat. Appln. Publn.
2016/0244787, both of which are hereby incorporated by reference in
their entireties and for all purposes) operatively linked to the
lac promoter under control of the lacI repressor. The expression
vector also contains the P15a origin of replication and a
chloramphenicol resistance gene. The resulting plasmids were
transformed into E. coli W3110, using standard methods known in the
art. The transformants were isolated by subjecting the cells to
chloramphenicol selection, as known in the art (See e.g., U.S. Pat.
No. 8,383,346 and WO2010/144103, both of which are incorporated by
reference herein, in their entirety).
Example 5
Preparation of HTP TG-Containing Wet Cell Pellets
E. coli cells containing recombinant TG-encoding genes from
monoclonal colonies were inoculated into the wells of 96 well
shallow-well microtiter plates containing 180 .mu.l LB containing
1% glucose and 30 .mu.g/mL chloramphenicol in each well. The plates
were sealed with O.sub.2-permeable seals and cultures were grown
overnight at 30.degree. C., 200 rpm and 85% humidity. Then, 10
.mu.l of each of the cell cultures were transferred into the wells
of 96-well deep-well plates containing 390 mL TB and 30 .mu.g/mL
CAM. The deep-well plates were sealed with O.sub.2-permeable seals
and incubated at 30.degree. C., 250 rpm and 85% humidity until an
OD.sub.600 of 0.6-0.8 was reached. The cell cultures were then
induced by IPTG to a final concentration of 1 mM and incubated
overnight under the same conditions as originally used. The cells
were then pelleted using centrifugation at 4000 rpm for 10 min. The
supernatants were discarded and the pellets frozen at -80.degree.
C. prior to lysis.
To lyse the cells, 225 .mu.l lysis buffer containing 20 mM Tris-HCl
buffer, pH 7.5, 1 mg/mL lysozyme, and 0.5 mg/mL PMBS was added to
the cell paste. The cells were incubated at room temperature for 2
hours with shaking on a bench top shaker. The plate was then
centrifuged for 15 minutes at 4000 rpm and 4.degree. C. and the
clear supernatants were used in subsequent steps.
To activate the pro-enzyme to the mature enzyme, 2 mg/mL of dispase
in 60 uL of 50 mM Tris-HCl buffer, pH 8.0 was added to 175 uL of
above E. coli supernatant and incubated for 1 hour at 37.degree.
C.
Example 6
HTP Purification of TG Variants
HTP purification of the activated lysate was carried out in
HisPur.TM. Ni-NTA spin plate (Life Technologies, cat #88230) using
manufacturer's protocol, with modifications, as described. First,
225 uL of dispase activated lysate obtained as described in Example
5 was diluted by an equal volume of binding buffer containing 50 mM
Na phosphate, pH 7.5, 300 mM NaCl, and 10 mM imidazole. Then, 165
uL of the diluted lysate was applied to HisPur.TM. Ni-NTA spin
plate pre-equilibrated in the binding buffer and incubated for 10
min at room temperature followed by centrifugation. This step was
repeated once. The spin plate was then washed with 600 uL of
washing buffer composed of 50 mM Na phosphate, pH 7.5, 300 mM NaCl.
and 25 mM imidazole. The purified enzyme was then eluted in 105 uL
of elution buffer containing 50 mM Na phosphate, pH 7.5, 300 mM
NaCl, and 250 mM imidazole.
Example 7
Preparation of Lyophilized Lysates from Shake Flask (SF)
Cultures
Selected HTP cultures grown as described above were plated onto LB
agar plates with 1% glucose and 30 .mu.g/ml CAM, and grown
overnight at 37.degree. C. A single colony from each culture was
transferred to 6 ml of LB with 1% glucose and 30 .mu.g/ml CAM. The
cultures were grown for 18 h at 30.degree. C., 250 rpm, and
subcultured approximately 1:50 into 250 ml of TB containing 30
.mu.g/ml CAM, to a final OD.sub.600 of 0.05. The cultures were
grown for approximately 195 minutes at 30.degree. C., 250 rpm, to
an OD.sub.600 between 0.6-0.8 and induced with 1 mM IPTG. The
cultures were then grown for 20 h at 30.degree. C., 250 rpm. The
cultures were centrifuged 4000 rpm.times.20 min. The supernatant
was discarded, and the pellets were resuspended in 30 ml of 20 mM
Tris-HCl, pH 7.5. The cells were pelleted (4000 rpm.times.20 min)
and frozen at -80.degree. C. for 120 minutes. Frozen pellets were
resuspended in 30 ml of 20 mM TRIS-HCl pH 7.5, and lysed using a
Microfluidizer.RTM. processor system (Microfluidics) at 18,000 psi.
The lysates were pelleted (10,000 rpm.times.60 min) and the
supernatants were frozen and lyophilized to generate shake flake
(SF) enzymes.
Example 8
Improvements in Activity of Transglutaminase Expressed by B.
megaterium
HTP B. megaterium cell pellets obtained as described in Example 3
were centrifuged for 15 minutes at 4000 rpm and 4.degree. C. and
the clear media supernatants were used in subsequent biocatalytic
reactions. HTP reactions were carried out in 96 well deep well
plates containing 100 .mu.L of 0.2 M Tris-HCl. pH 8.0, 0.04 M
glutamyl donor substrate Z-Gln-Gly (Sigma, C6154), 0.1 M
hydroxylamine, 0.01 M glutathione, and 5 .mu.l HTP B. megaterium
culture lysate supernatant. The HTP plates were incubated in a
Thermotron.RTM. titre-plate shaker (3 mm throw, model # AJ185,
Infors) at 37.degree. C., 100 rpm, for 35 min. The reactions were
quenched with 100 .mu.l 0.8 M HCl containing 0.3 M trichloraacetic
acid and 2 M FeCl.sub.3.6H.sub.2O. Absorbance of the samples was
recorded at 525 nm.
The fold improvement over positive control (FIOPC) was calculated
as the absorbance of the product normalized by the absorbance of
the corresponding backbone under the specified reaction conditions.
The results are shown in Table 8.1, below.
TABLE-US-00002 TABLE 8.1 Transglutaminase HTP Activity Results SEQ
ID NO: Amino Acid Differences Activity Improvement (nt/aa)
(Relative to SEQ ID NO: 6) (FIOP).sup..dagger. on Glutathione 7/8
G327R ++ 9/10 Y101G/Q201K/R212K/S287G ++ 11/12 Y101G/S287G ++ 13/14
S79K ++ 15/16 Y101G/Q201K/R285Q ++ 17/18 Y101G ++
.sup..dagger.Levels of increased activity or selectivity were
determined relative to the reference polypeptide of SEQ ID NO: 6,
and defined as follows: "+" > than 1.2-fold but less than
1.5-fold increase; "++" > than 1.5-fold but less than 2-fold;
"+++" > than 2-fold.
Example 9
Improvements in Activity Trasglutaminase Expressed in E. coli
Libraries of the parent enzyme (SEQ ID NO: 2) containing engineered
genes were produced using well established techniques known in the
art (e.g. saturation mutagenesis and recombination of previously
identified beneficial mutations). The polypeptides encoded by each
gene were produced in HTP as described in Example 4 and the soluble
lysate was generated as described in Example 5.
The following assays were used to evaluate the activity of these
variant polypeptides.
Assay A: Glutathione Assay
HTP reactions were carried out in 96-well deep-well plates
containing 100 .mu.L of 0.2 M Tris-HCl. pH 8.0, 0.04 M Z-Gln-Gly,
0.1 M hydroxylamine, 0.01 M glutathione, and 10 uL of activated
lysate supernatant. The HTP plates were incubated in a Thermotron
titre-plate shaker (3 mm throw, model # AJ185, Infors) at
37.degree. C., 300 rpm, for 30 min. The reactions were quenched
with 100 .mu.l of quenching solution containing 0.8 M HCl, 0.3 M
trichloroacetic acetate, and 2 M FeCl.sub.3.6H.sub.2O, mixed for 10
minutes using a bench top shaker. The plates were then centrifuged
at 4000 rpm for 5 minutes and absorbance at 525 nm recorded. The
fold improvement over positive control (FIOPC) was calculated as
the UV signal of the variants normalized by that of the
corresponding backbone under the specified reaction conditions.
Assay B: Insulin Assay
HTP reactions were carried out in 96-well deep-well plates
containing 200 .mu.L of 0.1 M Tris-HCl, pH 8.0, 1 g/L insulin, 25
mM EDTA, 1.25 mM Z-Gin-donor substrate, and 70 uL of purified
lysate as described in Example 6. The HTP plates were incubated in
a Thermotron.RTM. titre-plate shaker (3 mm throw, model # AJ185,
Infors) at 30.degree. C., 300 rpm, for 24 hours. The reactions were
quenched with 200 .mu.l DMSO and mixed for 5 minutes using a bench
top shaker. The plates were then centrifuged at 4000 rpm for 5
minutes, and the supernatants loaded into LC-MS for analysis. The
LC-MS and UV signals were both collected. The fold improvement over
positive control (FIOPC) was calculated as the UV signal of the
modified insulin in variants normalized by that of the
corresponding backbone under the specified reaction conditions.
TABLE-US-00003 TABLE 9.1 Assay Results for Transglutaminase
Variants Activity Activity Improvement Improvement SEQ ID
(FIOP).sup..dagger. on (FIOP).sup..dagger-dbl. on NO: Amino Acid
Differences Glutatione Insulin (nt/aa) (Relative to SEQ ID NO: 2)
(Assay A) (Assay B) 19/20 S48K/G203L/G296L/N343R/ ++++ E346H/K373M
21/22 S48K/Q170K/G203L/E346H/ ++ ++++ K373M 23/24
S48K/Q170K/G203L/R254Q/ ++ ++++ E346H 25/26 S48K/G203L/N343R/E346H/
+ ++++ K373M 27/28 S48K/G203L/R254Q/E346H/ +++ ++++ K373M 29/30
S48K/Q170K/G203L/N343R/ ++++ E346H 31/32 S48K/Q170K/G203L/G296L/
++++ N343R/E346H 33/34 S48K/G203L/N343R/E346H ++++ 35/36
N343R/E346H/K373M ++++ 37/38 S48K/Q170K/G203L/G296L/ +++ ++++
E346H/K373M 39/40 S48K/G203L/G296L/E346H/ +++ ++++ K373M 41/42
S48K/Q170K/G203L/R254Q/ +++ ++++ G296L/E346H/K373M 43/44
S48K/G203L/E346H ++ ++++ 45/46 S48K/Q170K/G203L/R254Q/ ++++
E346H/K373M 47/48 S48K/G203L/E346H/K373M ++++ 49/50
S48K/Q170K/G203L/R254Q/ ++ ++++ G296L/E346H 51/52 G203L/N343R/E346H
++++ 53/54 S48K/Q170K/G296L/N343R/ ++++ E346H 55/56
S48K/G203L/R254Q/E346H ++ ++++ 57/58 S48K/G203L/R254Q/G296L/ +++
++++ E346H/K373M 59/60 S48K/Q170K/G203L/E346H ++ ++++ 61/62
S48K/G203L/G296L/E346H ++++ 63/64 S48K/N343R/E346H ++++ 65/66
S48K/R254Q/E346H ++++ 67/68 S48V/R67E/G203V/S256G/ +++ +++
G296R/K373V 69/70 S48K/Q170K/N343R/E346H +++ 71/72
Q170K/G203L/N343R/E346H +++ 73/74 Q170K/G203L/R254Q/G296L/ ++ +++
N343R/E346H 75/76 G203L/E346H +++ 77/78 S48V/R67E/Y70G/S256G/ +++
+++ G296R/S345E/K373V 79/80 S48K/G203L/R254Q/G296L/ +++ +++
N343R/K373M 81/82 F297W/E346A +++ 83/84 S48V/S256G/G296R ++ +++
85/86 P68A/R282K/F297W/E346A +++ 87/88 F136Y/P215N/H234Y/F297W/ +++
E346A 89/90 P68A/E74T/S190G/P215N/ +++ E346A 91/92 E74T/F136Y/E346A
+++ 93/94 P215N/H234Y/F297W/E346A +++ 95/96 G203L/N343R ++ ++ 97/98
S48V/R67E/Y70G/G203V/ +++ ++ S256G/G296R/S345E 99/100
F136Y/S190G/P215N/F297W/ ++ E346A 101/102 E74T/E346A ++ 103/104
Q170K/G203L/R254Q/N343R/ ++ ++ K373M 105/106 S48V/G296R/K373V ++ ++
107/108 P68A/F297W/E346A ++ 109/110 P68A/V158I/E174D/H234Y/ ++
R282K/F297W/E346A 111/112 E174D/R282K/F297W/E346A ++ 113/114
E74T/F136Y/E174D/F297W/ ++ E346A 115/116 E74T/S255R/E346A ++
117/118 E174D/P215N/H234Y/F297W/ ++ E346A 119/120
S48V/R67E/Y70G/R181K/ +++ ++ G296R/S345E/K373V 121/122
P215H/S255R/F297W/E346A ++ 123/124 P215N/S255R/F297W/E346A ++
125/126 S48K/G203L/G296L/N343R/ +++ ++ K373M 127/128
S48V/R181K/G296R + ++ 129/130 P68A/F136Y/P215N/F297W/ ++ E346A
131/132 P215N/E346A ++ 133/134 S190G/F297W/E346A ++ 135/136
F136Y/F297W/E346A ++ 137/138 E174D/S190G/H234Y/F297W/ ++ E346A
139/140 S48V/R67E/Y70G/R181K/ +++ ++ G203V/S256G 141/142
E74T/F136Y/E174D/R282K/ ++ E346A 143/144 S255R/F297W/E346A ++
145/146 F136Y/E174D/P215N/S255R/ ++ R282K/F297W/E346A 147/148
V158I/P215N/S255R/E346A ++ ++ 149/150 P215N/F297W/E346A ++ 151/152
P68A/V158I/P215N/F297W/ ++ E346A 153/154 F136Y/V158I/P215N/F297W/
++ E346A 155/156 H234Y/S255R/E346A ++ 157/158 S255R/E346A ++
159/160 E346A ++ 161/162 F136Y/V158I/S190G/P215N/ ++
S255R/F297W/E346A 163/164 F136Y/P215N/F297W ++ 165/166
P68A/F136Y/P215N/S255R/ ++ R282K/F297W/E346A 167/168
P68A/P215N/F297W/E346A ++ 169/170 S48K/Y70L/G203L/R254Q/ +++ ++
G296L/N343R 171/172 S48V/G203V/S256G ++ ++ 173/174
S190G/S255R/R282K/E346A ++ 175/176 S48K/G203L/R254Q/G296L +++ ++
177/178 S48V/R67E/G203V/S256G/ ++ ++ S345E 179/180
V158I/P215N/E346A ++ 181/182 S48V/G203V/S256G/G296R/ ++ S345E
183/184 S48V/Y70N/G203V/K373V ++ ++ 185/186 S48V/Y70G/G203V/S256G/
+++ ++ S345E/K373V 187/188 S48V/R67E/Y70G ++ ++ 189/190 S48K/G203L
++ ++ 191/192 E174D/P215N/S255R/F297W/ ++ E346A 193/194
S48V/R181K/S256G/G296R/ ++ S345E 195/196 S48V/R67E/Y70G/G203V/S345E
++ ++ 197/198 S48V/R67E/Y70G/R181K/ ++ ++ S256G/S345E 199/200
S48V/R67E/Y70N/S256G ++ ++ 201/202 S48K/Q170K/G203L/K373M ++ ++
203/204 S48V/G203V ++ 205/206 S48V/R181K/G296R/S345E ++ 207/208
R67E/G296R/S345E ++ ++ 209/210 S48V ++ 211/212
S48V/S256G/G296R/S345E ++ 213/214 S48V/R67E/Y70N/G203V/ +++ ++
S256G/S345E/G354H/K373L 215/216 S48K/Q170K/G203L + ++ 217/218
F136Y/P215N/H234Y/R282K/ ++ F297W 219/220 S48V/R181K ++ 221/222
S48K/R254Q/G296L ++ ++ 223/224 R67E/S256G + ++ 225/226
S48K/Q170K/G296L + + 227/228 E74T/V158I/S255R/F297W + 229/230
S48V/G296R/S345E + 231/232 S48V/S256G + 233/234 P68A/H234Y ++ +
235/236 S48V/R181K/G203V/S256G/ ++ + S345E 237/238 S48K/Q170K/R254Q
+ 239/240 S48K/Y70D/Q170K/G203L ++ + 241/242 S48V/G203V/S345E +
243/244 G203L/G296L ++ + 245/246 P68A/F136Y/H234Y + 247/248
S48V/S345E/K373L + 249/250 S48V/Y70N/G203V/S256G/S345E ++ + 251/252
P215N/F297W + 253/254 S48V/R181K/G203V/S345E + .sup..dagger.Levels
of increased activity or selectivity were determined relative to
the reference polypeptide of SEQ ID NO: 2. and defined as follows:
"+" > than 1.2-fold but less than 1.5-fold increase: "++" >
than 1.5-fold but less than 2-fold; "+++" > than 2-fold.
.sup..dagger-dbl.Levels of increased activity or selectivity were
determined relative to the reference polypeptide of SEQ ID NO: 2,
and defined as follows: "+" > than 1.2-fold but less than
2.0-fold increase: "++" > than 2.0-fold but less than 5-fold:
"+++" > than 5-fold but less than 10-fold; "++++" > than
10-fold.
TABLE-US-00004 TABLE 9.2 Transglutaminase Variant Activity Results
Activity Activity Improvement Improvement SEQ ID
(FTOP).sup..dagger. on (FIOP).sup..dagger-dbl. on NO: Amino Acid
Differences Glutathione Insulin (nt/aa) (Relative to SEQ ID NO: 2)
(Assay A) (Assay B) 493/494 S48K/G203L/R254Q/N343R + ++ 495/496
S48V/R67E/G203V/S256G/ ++ ++ G296R/K373V/G378D 497/498
S48V/R67E/G203V/G296R/ ++ ++ K373V 499/500 S48V/Y70G/G203V/G296R/
++ ++ K373V 501/502 S48V/Y70G/G203V/S256G/ ++ ++ G296R/K373V
503/504 S48V/G203V/S256G/G296R/ ++ ++ K373V 505/506
S48V/R67E/S256G/G296R/ ++ ++ K373V 507/508 S48V/G203V/G296R/K373V/
+ ++ Q374L 509/510 S48V/R67E/Y70G/G203V/ ++ ++ S256G/G296R/K373V
511/512 S48V/G203V/G296R/K373V ++ ++ 513/514 S48V/R67E/Y70G/R181K/
++ ++ G203V/S256G/G296R/K373V 515/516 S48V/R67E/R181K/G203V/ ++ ++
S256G/G296R/K373V 517/518 S48V/Y70G/S256G/G296R/ ++ ++ K373V
519/520 S48V/Y70G/R181K/G203V/ ++ ++ S256G/G296R/K373V 521/522
S48V/G203V/S256G/G296R ++ ++ 523/524 A36E/S48K/G203L/R254Q/ ++ ++
E346H 525/526 S48V/Y70G/G203V/G296R ++ ++ 527/528
S48V/R181K/G203V/G296R + ++ 529/530 S48V/S256G/G296R/K373V + ++
531/532 S48V/R181K/S256G/G296R/ + ++ K373V 533/534
S48V/Y70G/R181K/G203V/ ++ ++ G296R/K373V 535/536
S48V/R181K/G203V/S256G/ ++ ++ K373V 537/538 G203V/S256G/G296R/K373V
++ ++ 539/540 S48V/R67E/R181K/S256G/ + ++ G296R 541/542
G203L/R254Q/N343R/E346H/ + ++ K373M 543/544 Y70G/G203V/S256G/G296R/
++ ++ K373V 545/546 R67E/R181K/G203V/S256G/ ++ ++ G296R/K373V
547/548 S48V/Y70G/G296R/K373V + ++ 549/550 R67E/Y70G/R181K/G203V/
++ ++ S256G/G296R/K373V 551/552 Y70G/R181K/G203V/G296R/ ++ ++ K373V
553/554 S48V/Y70G/G203V/K373V ++ ++ 555/556 S48V/R67E/R181K/G203V/
++ ++ S256G/K373V 557/558 S48V/R67E/G203V/S256G/ ++ ++ K373V
559/560 S48V/Y70G/G203V/S256G/ ++ ++ K373V 561/562
R181K/G203V/S256G/G296R/ ++ ++ K373V 563/564
R67E/G203V/S256G/G296R/ ++ ++ K373V 565/566 G203V/G296R/K373V + ++
567/568 R67E/S256G/G296R/K373V + ++ 569/570 S48V/S256G/K373V + ++
571/572 R181K/G203V/G296R/K373V + ++ 573/574 S48V/G203V/K373V + ++
575/576 A33D/R67E/Y70G/R181K/ ++ ++ G203V/S256G/G296R/K373V 577/578
S48V/R181K/G203V/K373V + ++ 579/580 S48V/G203V/S256G/K373V ++ ++
581/582 G203L/R254Q/E346H/K373M + ++ 583/584
R67E/R181K/G203V/S256G/ ++ ++ G296R 585/586
G203V/S256G/G296R/K373V/ + + H386Y 587/588 G203L/R254Q/E346H ++ +
589/590 S256G/G296R + + 591/592 Y70G/R181K/G203V/S256G/ ++ +
G296R/K373V 593/594 R67E/T70G/R181K/S256G/ + + G296R/K373V 595/596
G203V/S256G/G296R + + 597/598 Y70G/G203V/G296R/K373V + + 599/600
R181K/S256G/G296R/K373V + + 601/602 S48V/Y70G/R181K/G203V/ ++ +
S256G/K373V 603/604 G203L/P224T/R254Q/K373M + + 605/606
G203V/S256G/K373V + + 607/608 S256G/G296R/K373V + + 609/610
S48K/G203L/R254Q/N343R/ + + E346H/K373M 611/612
R181K/G203V/S256G/G296R + + 613/614 S48K/R254Q/N343R/E346H/ + +
K373M 615/616 R67E/R181K/G203V/S256G/ + + K373V 617/618 S48V/K373V
+ 619/620 S256G/K373V + + 621/622 R254Q/E346H/K373M + + 623/624
S48K/G203L/R254Q/N343R/ ++ + K373M 625/626 S48K/G203L/N343R/K373M +
+ 627/628 R181K/G203V/S256G/K373V + + 629/630
G203V/N209Y/S256G/K373V + + 631/632 R181K/G203V/K373V + + 633/634
G203L/E346H/K373M + + 635/636 G203V/S256G + + 637/638
H234Y/R282K/E346A + + 639/640 Y70G/G203V/S256G/K373V ++ + 641/642
R181K/G203V/S256G + + 643/644 F136Y/P215N/R282K/E346A + + 645/646
G203V/K373V + + 647/648 R67E/Y70G/R181K/K373V + 649/650 R254Q/E346H
+ 651/652 Y70G/G203V + 653/654 S48K/G203L/R254Q/N343R/ +
A355T/K373M 655/656 S48K/R254Q/E346H/K373M + 657/658
G203V/S256G/G296R/H320Y/ + K373V 659/660 G203L/R254Q/N343R/K373M ++
661/662 S48K/R254Q/N343R/K373M + 663/664 S48K/G203L/R254Q/E346D/ +
K373M 665/666 G203L/K373M + 667/668 S48K/R254Q + 669/670 K373M +
671/672 G203L/R254Q/K373M ++ 673/674 S48K/G203L/R254Q ++ 675/676
S48K/G203L/K373M ++ 677/678 S48K/R254Q/K373M + 679/680 R254Q/K373M
+ 681/682 G203L/R254Q + 683/684 S48K/G203L/R254Q/K373M ++ 685/686
R254Q + 687/688 E74T/F136Y/H234Y/R282K/ ++ F297W/E346A 689/690
E74T/F136Y/P215N/H234Y/ ++ R282K/F297W/E346A 691/692
E74T/P215N/H234Y/R282K/ ++ F297W/E346A 693/694
E74T/F136Y/P215N/R282K/ ++ F297W/E346A 695/696
P215N/H234Y/R282K/F297W/ ++ E346A 697/698 E74T/F136Y/P215N/H234Y/
++ R282K/E346A 699/700 E74T/F136Y/P215N/H234Y/ ++ F297W/E346A
701/702 E74T/F136Y/R282K/F297W/ ++ E346A 703/704
E74T/F136Y/P215N/F297W/ ++ E346A 705/706 E74T/P215N/R282K/F297W/ ++
E346A 707/708 E74T/F136Y/P215N/H234Y/ + F297W/N343Y/E346A 709/710
N343R/K373M + 711/712 E74T/F136Y/P215N/H234Y/ + E346A 713/714
S48V/R181K/G203V/S256G/ + G296R/K373V 715/716
F136Y/P215N/H234Y/R282K/ + F297W/E346A 717/718 F136Y/H234Y/E346A +
719/720 E74T/P215N/E346A + 721/722 E74T/F136Y/P215N/E346A + 723/724
F136Y/H234Y/F297W/E346A + 725/726 F136Y/P215N/R282K/F297W/ + E346A
727/728 F136Y/P215N/E346A + 729/730 P215N/H234Y/R282K/E346A +
731/732 F136Y/P215N/F297W/E346A + 733/734 E74T/F136Y/H234Y/E346A +
735/736 R282K/F297W/E346A + 737/738 P215N/H234Y/E346A + 739/740
E74T/F136Y/P215N/R282K/ + E346A 741/742 E74T/F136Y/P215N/H234Y/ +
F297W 743/744 K373V + 745/746 R181K/G296R + 747/748
S48K/A176T/G203L/R254Q/ + E346H/K373M 749/750
F136Y/P215N/R282K/F297W + 751/752 F136Y/H234Y/F297W + 753/754
E74T/P215N + 755/756 F136Y/R282K/F297W + .sup..dagger-dbl.Levels of
increased activity or selectivity were determined relative to the
reference polypeptide of SEQ ID NO: 2. and defined as follows: "+"
> than 1.2-fold but less than 2.0-fold increase; "++" > than
2.0-fold but less than 5-fold; "+++" > than 5-fold but less than
10-
Example 10
Activity Improvement in Transglutaminase Variants Expressed in E.
coli
Libraries of the parent enzyme (SEQ ID NO: 34) containing
engineered genes were produced using well established techniques
known in the art (e.g. saturation mutagenesis and recombination of
previously identified beneficial mutations). The polypeptides
encoded by each gene were produced in HTP as described in Example
4, and the soluble lysate was generated as described in Example 5.
HTP reactions were carried out in 96-well deep-well plates
containing 200 .mu.L of 0.1 M Tris-HCl, pH 8.0, 1 g/L insulin, 25
mM EDTA, 5 mM lysine donor substrate, and 70 uL of purified lysate
as described in Example 6. The HTP plates were incubated in a
Thermotron.RTM. titre-plate shaker (3 mm throw, model # AJ185,
Infors) at 30.degree. C., 300 rpm, for 22 hours. The reactions were
quenched with 200 .mu.l DMSO and mixed for 5 minutes using a bench
top shaker. The plates were then centrifuged at 4000 rpm for 5
minutes, supernatant loaded into LC-MS for analysis. The fold
improvement over positive control (FIOPC) was calculated as the
mass of insulin modified with one lysine donor in variants
normalized by that of the corresponding backbone under the
specified reaction conditions.
TABLE-US-00005 TABLE 10.1 Transglutaminase Variant Activity SEQ ID
NO: Amino Acid Differences Activity Improvement (nt/aa) (Relative
to SEQ ID NO: 34) (FIOP).sup..dagger-dbl. on Insulin 255/256 D50R
++ 257/258 D50A ++ 259/260 L331H ++ 261/262 L331P + 263/264 D50Q ++
265/266 K48S/D49W ++ 267/268 L331V + 269/270 D50F + 271/272 S292R +
273/274 T291C + 275/276 S330Y + 277/278 L331R + 279/280 S330H +
281/282 D49Y + .sup..dagger-dbl.Levels of increased activity or
selectivity were determined relative to the reference polypeptide
of SEQ ID NO: 34, and defined as follows: "+" > than 1.2-fold
but less than 2.0-fold increase; "++" > than 2.0-fold but less
than 5-fold; "+++" > than 5-fold but less than 10-fold, "++++"
> than 10-fold.
Example 11
Activity Improvement in Transglutaminase Variants Expressed in E.
coli
Libraries of the parent enzyme (SEQ ID NO: 256) containing
engineered genes were produced using well established techniques
known in the art (e.g., saturation mutagenesis and recombination of
previously identified beneficial mutations). The polypeptides
encoded by each gene were produced in HTP as described in Example
4, and the soluble lysates were generated as described in Example
5.
HTP reactions were carried out in 96-well deep-well plates
containing 200 .mu.L of 0.1 M Tris-HCl, pH 8.0, 2 g/L insulin, 25
mM EDTA, 5 mM lysine donor substrate, 10% acetonitrile, and 70 uL
of purified lysate produced as described in Example 6. The HTP
plates were incubated in a Thermotron.RTM. titre-plate shaker (3 mm
throw, model # AJ185, Infors) at 30.degree. C., 300 rpm, for 22
hours. The reactions were quenched with 200 .mu.l DMSO and mixed
for 5 minutes using a bench top shaker. The plates were then
centrifuged at 4000 rpm for 5 minutes, and supernatants loaded into
LC-MS for analysis. The fold improvement over positive control
(FIOPC) was calculated as the mass of insulin modified with one
lysine donor in variants normalized by that of the corresponding
backbone under the specified reaction conditions.
TABLE-US-00006 TABLE 11.1 Transglutaminase Variant Assay Results
Activity SEQ ID Improvement NO: Amino Acid Differences
(FIOP).sup..dagger-dbl. on (nt/aa) (Relative to SEQ ID NO: 256)
Insulin 283/284 K48S/D49W/R50A/L331V +++ 285/286
K48S/D49Y/R50A/T291C/S292R/L331V +++ 287/288
K48V/L203V/H234Y/H346A/K373M +++ 289/290 K48S/D49W/R50A/S292R ++
291/292 K48V/L203V/H234Y/S256G/H346A/K373M ++ 293/294 L203V/K373M
++ 295/296 K48S/D49W/S330Y/L331V ++ 297/298
R67E/Y70G/E74T/P215H/H234Y/F297W/ ++ H346A/K373L 299/300
K48S/D49W/R50A/S349R ++ 301/302 R67E/F297W/H346A ++ 303/304
R67E/E74T/P215H/H346A/K373V ++ 305/306
R67E/Y70G/E74T/F136Y/L203V/P215H/ ++ S256G/H346A/K373M 307/308
R67E/P215H/H234Y/F297W/H346A/K373V ++ 309/310
K48V/R67E/E74T/H234Y/F297W/H346A/ ++ K373M 311/312
S292R/S330Y/L331P ++ 313/314 R67E/Y70G/E74T/P215H/S256G/K373M ++
315/316 K48S/D49G/R50A/S292R/L331P ++ 317/318 K48V/R67E/H346A/K373M
++ 319/320 R67E/E74T/P215H/S256G/F297W/H346A/ ++ K373L 321/322
R67E/Y70G/F136Y/L203V/F297W/H346A/ ++ K373M 323/324
R67E/Y70G/L203V/P215H/S256G/H346A/ ++ K373L 325/326
K48V/R67E/Y70G/H234Y/S256G/R282K/ ++ F297W/H346A 327/328
K48V/R67E/E74T/H346A ++ 329/330 N27S/K48V/R67E/Y70G/H346A/K373L ++
331/332 D49W/R50A/L331V ++ 333/334
R67E/Y70G/E74T/L203V/P215H/H234Y/ ++ H346A/K373V 335/336
N27S/K48V/R67E/Y70G/F136Y/L203V/ ++ P215H/S256G/R282K/H346A/K373V
337/338 R67E/F136Y/L203V/P215H/S256G/ ++ H346A/K373V 339/340
Y70G/E74T/L203V/P215H/H346A/K373V ++ 341/342 R67E/Y70G/P215H ++
343/344 K48V/R67E/Y70G/L203V/P215H/H234Y/ ++ S256G/H346A 345/346
K48V/R67E/L203V/H346A/K373M ++ 347/348
N27S/K48V/R67E/E74T/L203V/S256G/ ++ H346A/K373M 349/350
R67E/E74T/F136Y ++ 351/352 N27S/R67E/H234Y/G296R/K373M ++ 353/354
K48V/R67E/P215H/R282K/F297W/ ++ H346A/K373M 355/356
F136Y/H346A/K373M ++ 357/358 R67E/F136Y/L203V/S256G/H346A/ ++ K373M
359/360 F297W/K373M ++ 361/362 K48V/E74T/H234Y/S256G/F297W/ ++
H346A/K373V 363/364 K48V/Y70G/E74T/F297W/H346A/ ++ K373M 365/366
K48V/Y70G/P215H/H234Y/S256G/ ++ H346A/K373M 367/368
N27S/K48V/R67E/Y70G/E74T/ ++ H234Y/S256G/R282K/H346A/K373L 369/370
K48V/R67E/E74T/L203V/H234Y/ ++ S256G/R282K/H346A/K373M 371/372
R67E/Y70G/L203V/K373M ++ 373/374 R67E/L203V/F297W/H346A/K373M ++
375/376 R67E/E74T/S256G/H346A/K373M ++ 377/378 H234Y/H346A/K373M ++
379/380 K48V/Y70G/L203V/P215H/S256G/ ++ R282K/H346A/K373V 381/382
K48V/F136Y/S256G/H346A/K373M ++ 383/384 K48V/S256G/K373L ++ 385/386
K48V/P215H/H346A/K373M ++ 387/388 K48V/P215H/H234Y/H346A/K373V ++
389/390 K48V/R67E/Y70G/H346A ++ 391/392 K48S/D49Y/R50Q/S292R/L331V
++ 393/394 S292R/S330Y/L331V ++ 395/396 E295R/F297Y/A333P ++
397/398 D49W/R50A/L331V/S349R ++ 399/400
K48V/I203V/H234Y/S256G/F297W/ ++ H346A/K373V 401/402
D49G/R50A/S292R/L331V ++ 403/404 K48V/E74T/L203V/H234Y/S256G/ ++
H346A/K373V 405/406 S292R/S349R ++ 407/408
K48V/H234Y/S256G/G296R/H346A/ ++ K373M 409/410 L203V/H234Y/H346A ++
411/412 D49G/R50Q/S292R/L331V/S349R ++ 413/414 S292R/L331V/S349R ++
415/416 S292R ++ 417/418 K48V/L203V/G296R/K373M ++ 419/420
S330Y/L331P ++ 421/422 K48V/R67E/H234Y/S256G/F297W/ ++ H346A/K373V
423/424 K48A/P287S/S292K/F297Y ++ 425/426
K48V/H234Y/S256G/H346A/K373M ++ 427/428
K48V/R67E/H234Y/S256G/H346A/ ++ K373M 429/430
R67E/E74T/L203V/H234Y/S256G ++ 431/432 L203V/H234Y/H346A/K373V ++
433/434 R67E/L203V/H234Y/S256G/H346A/ ++ K373V 435/436 R50A +
437/438 A45S/S292K/N328E + 439/440 S292R/L331V + 441/442
N328E/A333P + 443/444 L331V/S349R + 445/446 A333P + 447/448 L331V +
449/450 K48A/P287S/F297Y/N328E/A333P + 451/452 K373V + 453/454
H346A/K373V + 455/456 K48A + 457/458 K48A/S292K + 459/460 P287S +
461/462 K48A/R284G/S292K/A333P + 463/464 A45S/P287S/N328E/A333P +
465/466 P287S/E295R/F297Y + 467/468 P287S/S292K/F297Y + 469/470
S292K + 471/472 F136Y + 473/474 E295R + 475/476 K48A/S292K/F297Y +
477/478 S292K/F297Y + 479/480 F297Y/N328E + 481/482
P287S/S292K/E295R/F297Y + 483/484 P287S/S330G/A333P + 485/486
P287S/S292K + 487/488 K373M + 489/490 H234Y/R282K + 491/492 S330Y +
.sup..dagger-dbl.Levels of increased activity or selectivity were
determined relative to the reference polypeptide of SEQ ID NO: 256,
and defined as follows: "+" > than 1.2-fold but less than
2.0-fold increase; "++" > than 2.0-fold but less than 5-fold,
"+++" > than 5-fold but less than 10-fold: "++++" > than
10-fold.
Example 12
Analytical Detection of TG Production Formation
Data described in Examples 8-11 were collected using analytical
methods in Tables 12.1 or 12.2. LC-MS analysis methods for the
product resulting in the modification of insulin by the glutamine
Z-donor are provided in Table 12.1. The HTP assay mixtures prepared
and formation of the modified insulin product compound detected by
LC-MS-UV using the instrumental parameters and conditions shown in
Table 12.1. The mass of the product was used to determine the
substrate and product peaks and the UV signal was used to quantify
each species and compare to the positive control and calculate
FIOP.
TABLE-US-00007 TABLE 12.1 Analytical Method Instrument Thermo LXQ
Column Waters X-bridge C18 column: 50 .times. 3.0 mm, 5 um, with
Phenomenex C18 guard Cartridge: 5 .times. 3.0 mm, 5 .mu.m Mobile
Gradient (A: 0.2% formic acid in water; B: 0.2% Phase formic acid
in MeCN) Time(min) % A 0.0 75 1.0 75 4.0 70 5.0 5.0 6.0 75 7.0 75
Flow Rate 0.7 mL/min Run Time 7 min Column 45.degree. C.
Temperature Injection 10 .mu.L Volume MS LXQ; divert flow from MS
between 0-0.5 min. Detection BP extracted ions for: insulin product
(+6, +5, +4 species) = 969.2, 1163.0, 1453.0 modified insulin
product (+5. +4 species) = 1227.0, 1533.0 MS MS Polarity: Positive;
Ionization: ESI; Mode: Q1 Scan Conditions from 200-2000; Source
voltage: 5; Sheath gas: 60; Aux gas: 15; Cap temp: 350; Cap V: 35;
Tube lens: 110. UV UV 280 nm Detection Detector: PDA (Thermo LXQ);
Wavelength Step = 1 nm; Filter rise time = 1 sec; Sample rate = 5
Hz; Filter bandwidth = 1 nm Retention Insulin product at 3.1 min;
modified insulin product time at 4.6 min
LC-MS Analysis for the product resulting in the modification of
insulin by the lysine donor substrate is shown in Table 12.2. The
HTP assay mixtures prepared and formation of the modified insulin
product compound detected by LC-MS-UV using the instrumental
parameters and conditions are provided in Table 12.2. The mass of
the product was used to determine the substrate and product peaks
and the UV signal was used to quantify each species and compare to
the positive control and calculate FIOP.
TABLE-US-00008 TABLE 12.2 Analytical Method Instrument Thermo LXQ
Column Waters X-bridge C18 column: 50 .times. 3.0 mm, 5 um, with
Phenomenex C18 guard Cartridge: 5 .times. 3.0 mm, 5 .mu.m Mobile
Gradient (A: 0.2% formic acid in water; B: 0.2% Phase formic acid
in MeCN) Time(min) % A 0.0 75 1.0 75 4.0 70 5.0 5.0 6.0 75 7.0 75
Flow Rate 0.7 mL/min Run Time 7 min Column 45.degree. C.
Temperature Injection 10 .mu.L Volume MS LXQ; divert flow from MS
between 0-0.5 min. Detection BP extracted ions for: insulin product
(+6, +5, +4 species) = 969.3, 1162.8, 1453.0 mono-lysine modified
insulin product (+6, +5, +4 species) = 990.8, 1188.6, 1485.3
di-lysine modified insulin product (+6, +5, +4 species) = 1012.3,
1214.4, 1517.5 tri-lysine modified insulin product (+6, +5, +4
species)= 1033.7, 1239.9, 1549.5 MS MS Polarity: Positive;
Ionization: ESI; Mode: QI Scan Conditions from 200-2000; Source
voltage: 5; Sheath gas: 60; Aux gas: 15; Cap temp: 350; Cap V: 35;
Tube lens: 110 Retention Insulin product at 3.0 min; mono-lysine
modified time insulin product at 2.7 mm; di-lysine modified insulin
product at 2.5 min; tri-lysine modified insulin at 2.4 min
While the invention has been described with reference to the
specific embodiments, various changes can be made and equivalents
can be substituted to adapt to a particular situation, material,
composition of matter, process, process step or steps, thereby
achieving benefits of the invention without departing from the
scope of what is claimed.
For all purposes in the United States of America, each and every
publication and patent document cited in this disclosure is
incorporated herein by reference as if each such publication or
document was specifically and individually indicated to be
incorporated herein by reference. Citation of publications and
patent documents is not intended as an indication that any such
document is pertinent prior art, nor does it constitute an
admission as to its contents or date.
SEQUENCE LISTINGS
0 SQTB SEQUENCE LISTING The patent contains a lengthy "Sequence
Listing" section. A copy of the "Sequence Listing" is available in
electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US11319531B2-
). An electronic copy of the "Sequence Listing" will also be
available from the USPTO upon request and payment of the fee set
forth in 37 CFR 1.19(b)(3).
* * * * *
References